Stacked herbicide tolerance event 8264.44.06.1, related transgenic soybean lines, and detection thereof

ABSTRACT

This invention relates in part to soybean event pDAB8264.44.06.1 and includes a novel expression cassettes and transgenic inserts comprising multiple traits conferring resistance to glyphosate, aryloxyalkanoate, and glufosinate herbicides. This invention also relates in part to methods of controlling resistant weeds, plant breeding and herbicide tolerant plants. In some embodiments, the event sequence can be “stacked” with other traits, including, for example, other herbicide tolerance gene(s) and/or insect-inhibitory proteins. This invention further relates in part to endpoint TaqMan PCR assays for the detection of Event pDAB8264.44.06.1 in soybeans and related plant material. Some embodiments can perform high throughput zygosity analysis of plant material and other embodiments can be used to uniquely identify the zygosity of and breed soybean lines comprising the event of the subject invention. Kits and conditions useful in conducting these assays are also provided.

BACKGROUND OF THE INVENTION

Glyphosate (N-phosphonomethylglycine), a broad-spectrum herbicide,inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzymein the shikimate biosynthetic pathway that produces the essentialaromatic amino acids in plant cells. Inhibition of EPSPS effectivelydisrupts protein synthesis and thereby kills the affected plant cells.Because glyphosate is non-selective, it kills both weeds and cropplants. Thus it is useful with crop plants when one can modify the cropplants to be resistant to glyphosate, allowing the desirable plants tosurvive exposure to the glyphosate.

Recombinant DNA technology has been used to isolate mutant EPSPsynthases that are glyphosate-resistant. Such glyphosate-resistantmutant EPSP synthases can be transformed into plants and conferglyphosate-resistance upon the transformed plants. By way of example, aglyphosate tolerance gene was isolated from Agrobacterium strain CP4 asdescribed in U.S. Pat. No. 5,633,435. This reference and all referencescited herein are hereby incorporated by reference.

Other glyphosate tolerance genes have been created through theintroduction of mutations. These include the AroA gene isolated by Comaiand described at U.S. Pat. Nos. 5,094,945, 4,769,061 and 4,535,060. Asingle mutant has been utilized, as described in U.S. Pat. No.5,310,667, by substituting an alanine residue for a glycine residuebetween amino acid positions 80 and 120. Double mutants have beendescribed in U.S. Pat. Nos. 6,225,114 and 5,866,775 in which, inaddition to the above mutation, a second mutation (a threonine residuefor an alanine residue between positions 170 and 210) was introducedinto a wild-type EPSPS gene.

Other work resulted in the production of glyphosate resistant maizethrough the introduction of a modified maize EPSPS gene bearingmutations at residue 102 (changing threonine to isoleucine) and residue106 (changing proline to serine) of the amino acid sequence encoded byGenBank Accession No. X63374. See U.S. Pat. Nos. 6,566,587 and6,040,497.

Examples of events providing resistance to glyphosate in soybeansinclude soybean line GTS 40-3-2 (Padgette et al. 1995), soybean eventMON89788 (U.S. Pat. No. 7,608,761), U.S. Pat. No. 7,608,761 relates tosoybean event MON89788, each of which was produced by inserting the cp4epsps gene into soybean.

The widespread adoption of the glyphosate tolerant cropping system andthe increasing use of glyphosate has contributed to the prevalence ofglyphosate-resistant and difficult-to-control weeds in recent years. Inareas where growers are faced with glyphosate resistant weeds or a shiftto more difficult-to-control weed species, growers can compensate forglyphosate's weaknesses by tank mixing or alternating with otherherbicides that will control the missed weeds.

One popular and efficacious tankmix partner for controlling broadleafescapes in many instances has been 2,4-dichlorophenoxyacetic acid(2,4-D). 2,4-D, which has been used as a herbicide for more than 60years, provides broad spectrum, post-emergence control of many annual,biennial, and perennial broadleaf weeds including several key weeds incorn, soybeans, and cotton. Key weeds controlled by 2,4-D (560-1120 gae/ha rates) in row crop production include Ambrosia artemisiifolia,Ambrosia trifida, Xanthium strumarium, Chenopodium album, Helianthusannuus, Ipomoea sp., Abutilon theophrasti, Conyza Canadensis, and Sennaobtusifolia. 2,4-D provides partial control of several key weedsincluding Polygonum pensylvanicum, Polygonum persicaria, Cirsiumarvense, Taraxacum officinale, and Amaranthus sp. including Amaranthusrudis, and Amaranthus palmeri.

A limitation to further use of 2,4-D is that its selectivity in dicotcrops like soybean or cotton is very poor, and hence 2,4-D is nottypically used on (and generally not near) sensitive dicot crops.Additionally, 2,4-D's use in grass crops is somewhat limited by thenature of crop injury that can occur. 2,4-D in combination withglyphosate has been used to provide a more robust burndown treatmentprior to planting no-till soybeans and cotton; however, due to thesedicot species' sensitivity to 2,4-D, these burndown treatments mustoccur at least 14-30 days prior to planting (Agriliance, 2005).

One organism that has been extensively researched for its ability todegrade 2,4-D is Ralstonia eutropha, which contains a gene that codesfor tfdA (Streber et al., 1987), an enzyme which catalyzes the firststep in the mineralization pathway. (See U.S. Pat. No. 6,153,401 andGENBANK Acc. No. M16730). tfdA has been reported to degrade 2,4-D(Smejkal et al., 2001). The products that result from the degradationhave little to no herbicidal activity compared to 2,4-D. tfdA has beenused in transgenic plants to impart 2,4-D resistance in dicot plants(e.g., cotton and tobacco) normally sensitive to 2,4-D (Streber et al.(1989), Lyon et al. (1989), Lyon (1993), and U.S. Pat. No. 5,608,147).

A number of tfdA-type genes that encode proteins capable of degrading2,4-D have been identified from the environment and deposited into theGenbank database. Many homologues are similar to tfdA (>85% amino acididentity). However, there are a number of polynucleotide sequences thathave a significantly lower identity to tfdA (25-50%), yet have thecharacteristic residues associated with α-ketoglutarate dioxygenase Fe(II) dioxygenases.

An example of a 2,4-D-degrading gene with low sequence identity (<35%)to tfdA is the aad-12 gene from Delftia acidovorans (US Patent App2011/0203017). The aad-12 gene encodes an S-enantiomer-specificα-ketoglutarate-dependent dioxygenase which has been used in plants toconfer tolerance to certain phenoxy auxin herbicides, including, but notlimited to: phenoxyacetic acid herbicides such as 2,4-D and MCPA; andphenoxybutanoic acid herbicides, such as 2,4-DB and MCPB) andpyridyloxyalkanoic acid herbicides (e.g., pyridyloxyacetic acidherbicides such as triclopyr and fluroxypyr), and including acid, salt,or ester forms of the active ingredient(s). (See, e.g., WO 2007/053482).

Glufosinate-ammonium (“glufosinate”) is a non-systemic, non-selectiveherbicide in the phosphinothricin class of herbicides. Used primarilyfor post-emergence control of a wide range of broadleaf and grassyweeds, L-phosphinothricin, the active ingredient in glufosinate,controls weeds through the irreversible inhibition ofglutamine-synthase, an enzyme which is necessary for ammoniadetoxification in plants. Glufosinate herbicides are sold commercially,for example, under the brand names Ignite®, BASTA, and Liberty®.

The enzyme phosphinothricin N-acetyl transferase (PAT), isolated fromthe soil bacterium Streptomyces viridochromogenes, catalyzes theconversion of L-phosphinothricin to its inactive form by acetylation. Aplant-optimized form of the gene expressing PAT has been used insoybeans to confer tolerance to glufosinate herbicide. One such exampleof glufosinate resistant soybeans is event A5547-127. Most recently, theuse of glufosinate herbicide in combination with theglufosinate-tolerance trait has been proposed as a non-selective meansto effectively manage ALS- and glyphosate resistant weeds.

The expression of heterologous or foreign genes in plants is influencedby where the foreign gene is inserted in the chromosome. This could bedue to chromatin structure (e.g., heterochromatin) or the proximity oftranscriptional regulation elements (e.g., enhancers) close to theintegration site (Weising et al., Ann. Rev. Genet 22:421-477, 1988), forexample. The same gene in the same type of transgenic plant (or otherorganism) can exhibit a wide variation in expression level amongstdifferent events. There may also be differences in spatial or temporalpatterns of expression. For example, differences in the relativeexpression of a transgene in various plant tissues may not correspond tothe patterns expected from transcriptional regulatory elements presentin the introduced gene construct.

Thus, large numbers of events are often created and screened in order toidentify an event that expresses an introduced gene of interest to asatisfactory level for a given purpose. For commercial purposes, it iscommon to produce hundreds to thousands of different events and toscreen those events for a single event that has desired transgeneexpression levels and patterns. An event that has desired levels and/orpatterns of transgene expression is useful for introgressing thetransgene into other genetic backgrounds by sexual outcrossing usingconventional breeding methods. Progeny of such crosses maintain thetransgene expression characteristics of the original transformant. Thisstrategy is used to ensure reliable gene expression in a number ofvarieties that are well adapted to local growing conditions.

BRIEF SUMMARY OF THE INVENTION

The subject invention can provide, in part, effective means for managingweed resistance, which helps preserve the usefulness ofherbicide-tolerant technologies. The subject invention can also providegrowers with great flexibility and convenience in weed control options.

More specifically, the present invention relates in part to the soybean(Glycine max) event designated pDAB8264.44.06.1 (“EventpDAB8264.44.06.1”) having representative seed deposited with AmericanType Culture Collection (ATCC) with Accession No. PTA-11336, and progenyderived thereof. The subject invention includes soybean plantscomprising Event pDAB8264.44.06.1 (and includes soybean plantscomprising a transgenic insert in a genomic segment comprising SEQ IDNO:1 and SEQ ID NO:2).

The transgenic insert present in the subject event and deposited seedcomprises three herbicide tolerance genes: aad-12, 2mepsps, and a patgene. The aad-12 gene, derived from Delftia acidovorans, encodes thearyloxyalkanoate dioxygenase (AAD-12) protein, which confers toleranceto, e.g., 2,4-dichlorophenoxyacetic acid and pyridyloxyacetateherbicides. The 2mepsps gene, a modified EPSPS sequence isolated frommaize, produces a protein which confers tolerance to glyphosateherbicides. The pat gene, from the soil bacterium Streptomycesviridochromogenes, confers tolerance to the herbicide glufosinate.

Other aspects of the invention comprise progeny plants, soybeans, seeds,and/or regenerable parts of the plants and seeds and progeny comprisingsoybean event pDAB8264.44.06.1, as well as food or feed products madefrom any thereof. The invention also includes plant parts of EventpDAB8264.44.06.1 that include, but are not limited to, pollen, ovule,flowers, shoots, roots, leaves, nuclei of vegetative cells, pollencells, and other plant cells that comprise Event pDAB8264.44.06.1. Theinvention further relates to soybean plants having tolerance to multipleherbicides including phenoxyacetic acid herbicides, phenoxybutanoic acidherbicides, pyridyloxyalkanoic acid herbicides, glyphosate, and/orglufosinate. Such soybean plants may also be stacked with genes thatconfer tolerance to various other non-selective and selectiveherbicides, including but not limited to dicamba, imidazolinone, andHPPD herbicides. The invention further includes novel geneticcompositions Event pDAB8264.44.06.1 and aspects of agronomic performanceof soybean plants comprising Event pDAB8264.44.06.1.

This invention relates in part to plant breeding and herbicide tolerantplants. This invention includes a novel transformation event in soybeanplants comprising a polynucleotide, as described herein, inserted into aspecific site within the genome of a soybean cell.

In some embodiments, said event/polynucleotide can be “stacked” withother traits, including, for example, agronomic traits and/orinsect-inhibitory proteins. However, the subject invention includesplants having the single event, as described herein.

In some embodiments, the subject herbicide tolerance event can becombined in a breeding stack with an insect resistance event. In some ofthese embodiments, the insect resistance event comprises a cry1F geneand a cry1Ac gene. Some such events and stacks are specificallyexemplified herein, including soybean event 9582.812.9.1 (“the 812Event”) and soybean event 9582.814.19.1 (“the 814 Event”). Plants, plantcells, and seeds, for example, comprising any combination of the subjectevents are included in the subject invention. In some embodiments, thesubject invention includes the Soybean Event 9582.812.9.1 (812 Event),alone, as discussed in more detail below.

The additional traits may be stacked into the plant genome, or into thesame locus as Event pDAB8264.44.06.1, for example via plant breeding,re-transformation of the transgenic plant containing EventDAS-8264.44.06.1, or addition of new traits through targeted integrationvia homologous recombination.

Other embodiments include the excision of a portion or all of thetransgenic insert and/or flanking sequences of Event DAS-8264.44.06.1.Upon excision, another and/or additional insert can be targeted to thespecific chromosomal site of Event DAS-8264.44.06.1. The exemplifiedinsert can be replaced, or further insert(s) can be stacked, in thismanner, with the exemplified insert of the subject soybean event.

In one embodiment, the present invention encompasses a soybeanchromosomal target site located on chromosome 6. In some embodiments,the target site comprises a heterologous nucleic acid. In someembodiments, the soybean chromosomal target site is located between orwithin the genomic flanking sequences set forth in SEQ ID NO:1 and SEQID NO:2.

In one embodiment, the present invention encompasses a method of makinga transgenic soybean plant comprising inserting a heterologous nucleicacid at a position on chromosome 6. In another embodiment, theheterologous nucleic acid is inserted on chromosome 6 near or betweenvarious exemplified polynucleotide segments as described herein.

Additionally, the subject invention provides assays for detecting thepresence of the subject event in a sample (of soybeans, for example).The assays can be based on the DNA sequence of the recombinantconstruct, inserted into the soybean genome, and on the genomicsequences flanking the insertion site. Kits and conditions useful inconducting the assays are also provided.

Thus, the subject invention relates in part to the cloning and analysisof the DNA sequences of the whole exemplified insert and the borderregions thereof (in transgenic soybean lines). These sequences areunique. Based on these insert and border (and junction) sequences,event-specific primers can be and were generated. PCR analysisdemonstrated that the events can be identified by analysis of the PCRamplicons generated with these event-specific primer sets. Thus, theseand other related procedures can be used to uniquely identify soybeanlines comprising the event of the subject invention.

The subject invention also relates in part to realtime or endpointTaqMan PCR assays for the detection of event 8264.44.06.1. Someembodiments are directed to assays that are capable of high throughputzygosity analysis. The subject invention further relates, in part, tothe use of a GMFL01-25-J19 (GenBank: AK286292.1) reference gene for usein determining zygosity. These and other related procedures can be usedto uniquely identify the zygosity of Event pDAB8264.44.06.1 and breedsoybean lines comprising the event.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plasmid map of pDAB8264.

FIG. 2 is a schematic diagram depicting primer locations for soybeanEvent pDAB8264.44.06.1.

FIG. 3 is a schematic diagram depicting primer locations and genomic DNAdeletion in soybean Event pDAB8264.44.06.1.

FIG. 4 is a schematic diagram depicting primer locations for the TaqManassay detection of soybean Event pDAB8264.44.06.1.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 provides the 5′ flanking border sequence for the subjectsoybean Event pDAB8264.44.06.1.

SEQ ID NO:2 provides the 3′ flanking border sequence for the subjectsoybean Event pDAB8264.44.06.1.

SEQ ID NO:3 provides primer 4406_WF1.

SEQ ID NO:4 provides primer 4406_WF2.

SEQ ID NO:5 provides primer 4406_WF3.

SEQ ID NO:6 provides primer 4406_WF4.

SEQ ID NO:7 provides primer 4406_WR5.

SEQ ID NO:8 provides primer 4406_WR6.

SEQ ID NO:9 provides primer 4406_WR7.

SEQ ID NO:10 provides primer 4406_WR8.

SEQ ID NO:11 provides primer ED_v1_C1.

SEQ ID NO:12 provides primer PAT_12.

SEQ ID NO:13 provides sequence for plasmid pDAB8264.

SEQ ID NO:14 provides partial 5′ soybean genomic flanking and partial 5′insert sequence.

SEQ ID NO:15 provides partial 3′ soybean genomic flanking and partial 3′insert sequence.

SEQ ID NO:16 provides a 98 base pair sequence spanning the 5′integration junction.

SEQ ID NO:17 provides a 131 base pair sequence spanning the 3′integration junction.

SEQ ID NO:18 provides primer 4406_5′F.

SEQ ID NO:19 provides primer 4406_5′R.

SEQ ID NO:20 provides probe 4406_5′P.

SEQ ID NO:21 provides primer 4406_3′F.

SEQ ID NO:22 provides primer 4406_3′R.

SEQ ID NO:23 provides probe 4406_3′P.

SEQ ID NO:24 provides primer GMS116F.

SEQ ID NO:25 provides primer GMS116R.

SEQ ID NO:26 provides probe GMS116Probe SEQ ID NO:27 provides thesequence of soybean Event pDAB8264.44.06.1, including the 5′ genomicflanking sequence, insert, and 3′ genomic flanking sequence.

SEQ ID NO:28 provides the expected sequence of Soybean Event9582.812.9.1, including the 5′ genomic flanking sequence, pDAB9582T-strand insert, and 3′ genomic flanking sequence.

SEQ ID NO:29 provides the expected sequence of Soybean Event9582.814.19.1, including the 5′ genomic flanking sequence, pDAB9582T-strand insert, and 3′ genomic flanking sequence.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein includes novel transformation events ofsoybean plants (soybean) comprising a cassette for the expression ofmultiple herbicide tolerance genes inserted into a specific locus withinthe genome of a soybean cell.

The exemplified transgenic insert comprising Event pDAB8264.44.06.1includes genetic elements for the expression of three differentherbicide tolerance genes: (1) a synthetic aad-12 gene; (2) an EPSPSsequence from maize encoding a protein containing mutations, as comparedto the wild-type EPSPS polypeptide: at amino acid residues 102 (fromthreonine to isoleucine) and 106 (from proline to serine) and whichconfers resistance or tolerance to glyphosate herbicides; and (3) a patgene which confers tolerance or resistance to the glufosinateherbicides. The aad-12 gene was derived from Delftia acidovorans andencodes an aryloxyalkanoate dioxygenase (AAD-12) protein enzyme capableof deactivating herbicides having an α-ketoglutarate moiety, includingphenoxyalkanoate herbicides (e.g., phenoxyacetic acid herbicides such as2,4-D and MCPA; phenoxypropionic acid herbicides such as dichlorprop,mecoprop and their enantiomers; and phenoxybutanoic acid herbicides suchas 2,4-DB and MCPB) and pyridyloxyalkanoic acid herbicides (e.g.,pyridyloxyacetic acid herbicides such as triclopyr and fluroxypyr),including acid, salt, or ester forms of the active ingredient(s)

More specifically, the subject invention relates in part to transgenicsoybean Event pDAB8264.44.06.1, plant lines comprising these events, andthe cloning and analysis of the DNA sequences of this insert, and/or theborder regions thereof. Plant lines of the subject invention can bedetected using sequences disclosed and suggested herein.

This invention relates in part to plant breeding and herbicide tolerantplants. In some embodiments, said polynucleotide sequence can be“stacked” with other traits (such as other herbicide tolerance gene(s)and/or gene(s) that encode insect-inhibitory proteins or inhibitory RNAsequences, for example). However, the subject invention also includesplants having a single event, as described herein.

In some embodiments, the subject herbicide tolerance event can becombined in a breeding stack with an insect resistance event. In someembodiments, the insect resistance event is selected from the groupconsisting of the 812 Event and the 814 Event (as defined in greaterdetail below), each of which comprises a cry1F gene and a cry1Ac gene.Plants, plant cells, and seeds, for example, comprising any combinationof the subject events are included in the subject invention. The subjectinvention also includes the novel 812 Event, alone, in certainembodiments, including plants, plant cells, and seeds, for example.

U.S. provisional application Ser. No. 61/471,845, filed Apr. 5, 2011,relates in part to soybean lines comprising Soybean Event 9582.812.9.1(the 812 Event). Seeds comprising this event were deposited and madeavailable to the public without restriction (but subject to patentrights), with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va., 20110. The deposit, designated asATCC Deposit No. PTA-11602, was made on Jan. 20, 2011. This deposit wasmade and will be maintained in accordance with and under the terms ofthe Budapest Treaty with respect to seed deposits for the purposes ofpatent procedure.

U.S. provisional applications Ser. Nos. 61/511,664 (filed Jul. 26, 2011)and 61/521,798 (filed Aug. 10, 2011) relates in part to soybean linescomprising soybean event 9582.814.19.1 (the 814 Event). Seeds comprisingthis event were deposited with the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va., 20110. The deposit,ATCC Patent Deposit Designation PTA-12006, was received by the ATCC onJul. 21, 2011. This deposit was made and will be maintained inaccordance with and under the terms of the Budapest Treaty with respectto seed deposits for the purposes of patent procedure.

The subject invention also includes plants, seeds, and plant cells, forexample, comprising SEQ ID NO:27 (Event pDAB8264.44.06.1; the 4406Event), SEQ ID NO:28 (the 812 Event), and/or SEQ ID NO:29 (the 814Event), and variants of these sequences having, for example, at least95,%, 96%, 97%, 98%, or 99% identity with such sequences. It is notuncommon for some variation (such as deletion of some segments) to occurupon integration of an insert sequence within the plant genome. This isdiscussed in more detail in Example 7, for example.

The subject invention also provides assays for detecting the presence ofthe subject event in a sample. Aspects of the subject invention includemethods of designing and/or producing any diagnostic nucleic acidmolecules exemplified or suggested herein, particularly those basedwholly or partially on the subject flanking sequences.

In some embodiments, a polynucleotide segment exemplified or describedherein (such as SEQ ID NO:1, SEQ ID NO:2, and/or the inserttherebetween, as depicted in FIG. 2 for example) can be excised andsubsequently re-targeted with additional polynucleotide sequence(s).

In some embodiments, this invention relates to herbicide-tolerantsoybean lines, and the identification thereof. The subject inventionrelates in part to detecting the presence of the subject event in orderto determine whether progeny of a sexual cross contain the event ofinterest. In addition, a method for detecting the event is included andis helpful, for example, for complying with regulations requiring thepre-market approval and labeling of foods derived from recombinant cropplants, for example. It is possible to detect the presence of thesubject event by any well-known nucleic acid detection method such aspolymerase chain reaction (PCR) or DNA hybridization using nucleic acidprobes. Event-specific PCR assays are discussed herein. (See e.g.Windels et al. (Med. Fac. Landbouww, Univ. Gent 64/5b:459462, 1999) foranother example.) Some of these examples relate to using a primer setspanning the junction between the insert and flanking DNA. Morespecifically, one primer included sequence from the insert and a secondprimer included sequence from flanking DNA.

Exemplified herein is soybean Event pDAB8264.44.06.1, and its selectionand characterization for stability and expression in soybean plants fromgeneration to generation. Both flanking sequences of EventpDAB8264.44.06.1 have been sequenced and are described herein as SEQ IDNO:1 and SEQ ID NO:2. Event specific assays were developed. It has alsobeen mapped onto the soybean genome (soybean chromosome 6). EventpDAB8264.44.06.1 can be introgressed into elite cultivars where it willconfer tolerance to phenoxy auxin, glyphosate and glufosinate herbicidesin inbred and hybrid soybean lines.

The subject EPSPS gene encodes a mutant 5-enolpyruvyl-3-phosphoshikimicacid synthase (EPSPS). The wild-type EPSPS gene was originally isolatedfrom Zea mays, and the sequence was deposited under GenBank accessionnumber X63374. See also U.S. Pat. No. 6,566,587 (in particular, SEQ IDNo. 3 therein).

To obtain high expression of heterologous genes in plants, it may bepreferred to reengineer said genes so that they are more efficientlyexpressed in plant cells. Modification of the wild-type plant EPSPSnucleotide sequence can provide such resistance when expressed in aplant cell. As described in the '587 patent, when comparing an EPSPSpolypeptide to the wild-type polypeptide, modification to substituteisoleucine for threonine at residue 102 and substitute serine forproline at position 106 of the protein, the result is the double mutantEPSPS polypeptide (2mEPSPS) used in the subject insert. When expressedin a plant cell, it provides tolerance to glyphosate. The subject EPSPSgene, also referred to as the “2mepsps gene” or DMMG, can alternativelybe optimized to improve expression in both dicotyledonous plants as wellas monocotyledonous plants, and in particular in soybean. Codon usagecan be selected based upon preferred hemicot codon usage, i.e.redesigned such that the protein is encoded by codons having a biastoward both monocot and dicot plant usage. Deleterious sequences andsuperfluous restriction sites can be removed to increase the efficiencyof transcription/translation of the 2mepsps coding sequence and tofacilitate DNA manipulation steps. A hemicot-optimized version of thesubject monocot gene is further detailed in U.S. Ser. No. 13/303,502(filed Nov. 23, 2011, claiming priority to Dec. 3, 2010) entitled,“OPTIMIZED EXPRESSION OF GLYPHOSATE RESISTANCE ENCODING NUCLEIC ACIDMOLECULES IN PLANT CELLS.”

As previously referenced herein, the introduction and integration of atransgene into a plant genome involves some random events (hence thename “event” for a given insertion that is expressed). That is, withmany transformation techniques such as Agrobacterium transformation, the“gene gun,” and WHISKERS, it is unpredictable where in the genome atransgene will become inserted. Thus, identifying the flanking plantgenomic DNA on both sides of the insert can be important for identifyinga plant that has a given insertion event. For example, PCR primers canbe designed that generate a PCR amplicon across the junction region ofthe insert and the host genome. This PCR amplicon can be used toidentify a unique or distinct type of insertion event.

During the process of introducing an insert into the genome of plantcells, it is not uncommon for some deletions or other alterations of theinsert and/or genomic flanking sequences to occur. Thus, the relevantsegment of the plasmid sequence provided herein might comprise someminor variations. The same is true for the flanking sequences providedherein. Thus, a plant comprising a polynucleotide having some range ofidentity with the subject flanking and/or insert sequences is within thescope of the subject invention. Identity to the sequence of the presentinvention can be a polynucleotide sequence having at least 65% sequenceidentity, more preferably at least 70% sequence identity, morepreferably at least 75% sequence identity, more preferably at least 80%identity, and more preferably at least 85% 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with a sequenceexemplified or described herein. Hybridization and hybridizationconditions as provided herein can also be used to define such plants andpolynculeotide sequences of the subject invention. The sequence whichcomprises the flanking sequences plus the full insert sequence can beconfirmed with reference to the deposited seed.

As “events” are originally random events, as part of this disclosure atleast 2500 seeds of a soybean line comprising Event pDAB8264.44.06.1have been deposited and made available to the public without restriction(but subject to patent rights), with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va., 20110. Thedeposit has been designated as ATCC Deposit No. PTA-11336. 100 packets(25 seeds per packet) of Glycine max seeds (“Soybean Seed Glycine maxL.: pDAB8264.44.06.1”) were deposited on behalf of Dow AgroSciences LLCand MS Technologies, LLC on Sep. 14, 2010. The deposit was tested onOct. 4, 2010, and on that date, the seeds were viable. This deposit wasmade and will be maintained in accordance with and under the terms ofthe Budapest Treaty with respect to seed deposits for the purposes ofpatent procedure. The deposit will be maintained without restriction atthe ATCC depository, which is a public depository, for a period of 30years, or five years after the most recent request, or for the effectivelife of the patent, whichever is longer, and will be replaced if itbecomes nonviable during that period.

As part of this disclosure at least 2500 seeds of a soybean linecomprising Event pDAB9582.812.9.1 and Event pDAB8264.44.06.1 (thesubject herbicide tolerance event and the 812 insect resistance event)have been deposited and made available to the public without restriction(but subject to patent rights), with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va., 20110. Thedeposit has been identified as “Designation: pDAB9582.812.9.1::EventpDAB8264.44.06.1” by the ATCC. 100 packets (25 seeds per packet) ofGlycine max seeds (“Soybean Seed Glycine max L.: pDAB8264.44.06.1”) weredeposited on Nov. 18, 2011. This deposit was made and will be maintainedin accordance with and under the terms of the Budapest Treaty withrespect to seed deposits for the purposes of patent procedure. Thedeposit will be maintained without restriction at the ATCC depository,which is a public depository, for a period of 30 years, or five yearsafter the most recent request, or for the effective life of the patent,whichever is longer, and will be replaced if it becomes nonviable duringthat period.

The deposited seeds are part of the subject invention. Clearly, soybeanplants can be grown from these seeds, and such plants are part of thesubject invention. The subject invention also relates to DNA sequencescontained in these soybean plants that are useful for detecting theseplants and progeny thereof. Detection methods and kits of the subjectinvention can be directed to identifying any one, two, or even all threeof these events, depending on the ultimate purpose of the test.

Definitions and examples are provided herein to help describe thepresent invention and to guide those of ordinary skill in the art topractice the invention. Unless otherwise noted, terms are to beunderstood according to conventional usage by those of ordinary skill inthe relevant art. The nomenclature for DNA bases as set forth at 37 CFR§1.822 is used.

As used herein, the term “progeny” denotes the offspring of anygeneration of a parent plant which comprises soybean EventpDAB8264.44.06.1.

A transgenic “event” is produced by transformation of plant cells withheterologous DNA, i.e., a nucleic acid construct that includes atransgene of interest, regeneration of a population of plants resultingfrom the insertion of the transgene into the genome of the plant, andselection of a particular plant characterized by insertion into aparticular genome location. The term “event” refers to the originaltransformant and progeny of the transformant that include theheterologous DNA. The term “event” also refers to progeny produced by asexual outcross between the transformant and another variety thatincludes the genomic/transgene DNA. Even after repeated back-crossing toa recurrent parent, the inserted transgene DNA and flanking genomic DNA(genomic/transgene DNA) from the transformed parent is present in theprogeny of the cross at the same chromosomal location. The term “event”also refers to DNA from the original transformant and progeny thereofcomprising the inserted DNA and flanking genomic sequence immediatelyadjacent to the inserted DNA that would be expected to be transferred toa progeny that receives inserted DNA including the transgene of interestas the result of a sexual cross of one parental line that includes theinserted DNA (e.g., the original transformant and progeny resulting fromselfing) and a parental line that does not contain the inserted DNA.

A “junction sequence” spans the point at which DNA inserted into thegenome is linked to DNA from the soybean native genome flanking theinsertion point, the identification or detection of one or the otherjunction sequences in a plant's genetic material being sufficient to bediagnostic for the event. Included are the DNA sequences that span theinsertions in herein-described soybean events and similar lengths offlanking DNA. Specific examples of such diagnostic sequences areprovided herein; however, other sequences that overlap the junctions ofthe insertions, or the junctions of the insertions and the genomicsequence, are also diagnostic and could be used according to the subjectinvention.

The subject invention relates in part to event identification using suchflanking, junction, and insert sequences. Related PCR primers andamplicons are included in the invention. According to the subjectinvention, PCR analysis methods using amplicons that span acrossinserted DNA and its borders can be used to detect or identifycommercialized transgenic soybean varieties or lines derived from thesubject proprietary transgenic soybean lines.

The binary plasmid, pDAB8264 (SEQ ID NO:13) comprises the geneticelements depicted in FIG. 1. The following genetic elements (T-strandborder sequences are not included) are contained within the T-strandregion of pDAB8264. In Table 1, the residue numbering of the geneticelements is provided with respect to SEQ ID NO:13 disclosed herein.

TABLE 1 Residue Numbering of the Genetic Elements Comprising BinaryPlasmid pDAB8264 (SEQ ID NO: 13). Genetic Element Position Reference RB7MARv3 (Matrix  137 bp-1302 bp Thompson and Myatt, Attachment Region)(1997) Plant Mol. Biol., 34: 687-692.; WO9727207 Intervening Sequence1303 bp-1341 bp Not applicable Histone H4A7 48 1342 bp-2002 bp Chaboutéet al., (1987) 3′UTR (Untranslated Plant Mol. Biol., 8: Region) 179-191Intervening Sequence 2003 bp-2025 bp Not applicable 2mepsps v1 2026bp-3363 bp U.S. Pat. No. 6,566,587 OTPc (optimized 3364 bp-3735 bp U.S.Pat. No. 5,510,471 transit peptide) Intervening Sequence 3736 bp-3748 bpNot applicable Intron 2 3749 bp-4214 bp Chaubet et al., (1992) J. Mol.Biol., 225: 569-574 Histone H4A7 48 4215 bp-5169 bp Chabouté et al.,(1987) Promoter Plant Mol. Biol., 8: 179-191 Intervening Sequence 5170bp-5261 bp Not applicable AtUbi 10 Promoter 5262 bp-6583 bp Callis, etal., (1990) (Arabidopsis thaliana J. Biol. Chem., 265: Ubiquitin 1012486-12493 Promoter) Intervening Sequence 6584 bp-6591 bp Notapplicable aad-12 v1 6592 bp-7473 bp WO 2007/053482 Intervening Sequence7474 bp-7575 bp Not applicable containing stop codons in all 6-framesAtuORF23 3′ UTR 7576 bp-8032 bp U.S. Pat. No. 5,428,147 (Agrobacteriumtumefaciens Open Reading Frame 23 UTR) Intervening Sequence 8033 bp-8146bp Not applicable CsVMV Promoter 8147 bp-8663 bp Verdaguer et al.,(1996) (Cassava Vein Mosaic Plant Mol. Biol., 31: Virus Promoter)1129-1139 Intervening Sequence 8664 bp-8670 bp Not applicable pat v68671 bp-9222 bp Wohlleben et al., (1988) Gene 70: 25-37 InterveningSequence 9223 bp-9324 bp Not applicable containing stop codons in all6-frames AtuORF1 3′UTR  9325 bp-10028 bp Huang et al., (1990)(Agrobacterium J. Bacteriol. 172: tumefaciens Open 1814-1822 ReadingFrame 1 UTR)

SEQ ID NOs: 14 and 15, respectively, are the 5′ and 3′ flankingsequences together with 5′ and 3′ portions of the insert sequence, asdescribed in more detail below, and thus include the 5′ and 3′“junction” or “transition” sequences of the insert and the genomic DNA.With respect to SEQ ID NO:14, residues 1-570 are 5′ genomic flankingsequence, and residues 571-859 are residues of the 5′ end of the insert.With respect to SEQ ID NO:15, residues 1-220 are residues of the 3′ endof the insert, and residues 221-1719 are 3′ genomic flanking sequence.The junction sequence or transition with respect to the 5′ end of theinsert thus occurs at residues 570-571 of SEQ ID NO:14. The junctionsequence or transition with respect to the 3′ end of the insert thusoccurs at residues 220-221 of SEQ ID NO:15. Polynucleotides of thesubject invention include those comprising, for example, 5, 10, 20, 50,100, 150, or 200 bases, or possibly more, and any incrementstherebetween, on either side of the junction sequence. Thus, a primerspanning the junction sequence could comprise, for example, 5-10 basesthat would hybridize with flanking sequence and 5-10 bases that wouldhybridize with insert sequence. Probes and amplicons could be similarlydesigned, although they would often be longer than primers.

The subject sequences (including the flanking sequences) are unique.Based on these insert and flanking sequences, event-specific primerswere generated. PCR analysis demonstrated that these soybean lines canbe identified in different soybean genotypes by analysis of the PCRamplicons generated with these event-specific primer sets. Thus, theseand other related procedures can be used to uniquely identify thesesoybean lines. The sequences identified herein are unique.

Detection techniques of the subject invention are especially useful inconjunction with plant breeding, to determine which progeny plantscomprise a given event, after a parent plant comprising an event ofinterest is crossed with another plant line in an effort to impart oneor more additional traits of interest in the progeny. These PCR analysismethods benefit soybean breeding programs as well as quality control,especially for commercialized transgenic soybean seeds. PCR detectionkits for these transgenic soybean lines can also now be made and used.This can also benefit product registration and product stewardship.

Furthermore, flanking soybean genomic sequences can be used tospecifically identify the genomic location of each insert. Thisinformation can be used to make molecular marker systems specific toeach event. These can be used for accelerated breeding strategies and toestablish linkage data.

Still further, the flanking sequence information can be used to studyand characterize transgene integration processes, genomic integrationsite characteristics, event sorting, stability of transgenes and theirflanking sequences, and gene expression (especially related to genesilencing, transgene methylation patterns, position effects, andpotential expression-related elements such as MARS [matrix attachmentregions], and the like).

In light of the subject disclosure, it should be clear that the subjectinvention includes seeds available under ATCC Deposit No. PTA-11336. Thesubject invention also includes a herbicide-tolerant soybean plant grownfrom a seed deposited with the ATCC under accession number PTA-11336.The subject invention further includes parts of said plant, such asleaves, tissue samples, seeds produced by said plant, pollen, and thelike (wherein they comprise a transgenic insert flanked by SEQ ID NO:1and SEQ ID NO:2).

Still further, the subject invention includes descendant and/or progenyplants of plants grown from the deposited seed, preferably aherbicide-resistant soybean plant wherein said plant has a genomecomprising a detectable wild-type genomic DNA/insert DNA junctionsequence as described herein. As used herein, the term “soybean” meansGlycine max and includes all varieties thereof that can be bred with asoybean plant.

The invention further includes processes of making crosses using a plantof the subject invention as at least one parent. For example, thesubject invention includes an F₁ hybrid plant having as one or bothparents any of the plants exemplified herein. Also within the subjectinvention is seed produced by such F₁ hybrids of the subject invention.This invention includes a method for producing an F₁ hybrid seed bycrossing an exemplified plant with a different (e.g. in-bred parent)plant and harvesting the resultant hybrid seed. The subject inventionincludes an exemplified plant that is either a female parent or a maleparent. Characteristics of the resulting plants may be improved bycareful consideration of the parent plants.

A herbicide-tolerant soybean plant of the subject invention can be bredby first sexually crossing a first parental soybean plant consisting ofa soybean plant grown from seed of any one of the lines referred toherein, and a second parental soybean plant, thereby producing aplurality of first progeny plants; then selecting a first progeny plantthat is resistant to a herbicide (or that possesses at least one of theevents of the subject invention); selfing the first progeny plant,thereby producing a plurality of second progeny plants; and thenselecting from the second progeny plants a plant that is resistant to aherbicide (or that possesses at least one of the events of the subjectinvention). These steps can further include the back-crossing of thefirst progeny plant or the second progeny plant to the second parentalsoybean plant or a third parental soybean plant. A soybean cropcomprising soybean seeds of the subject invention, or progeny thereof,can then be planted.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating, added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Otherbreeding methods commonly used for different traits and crops are knownin the art. Backcross breeding has been used to transfer genes for asimply inherited, highly heritable trait into a desirable homozygouscultivar or inbred line, which is the recurrent parent. The source ofthe trait to be transferred is called the donor parent. The resultingplant is expected to have the attributes of the recurrent parent (e.g.,cultivar) and the desirable trait transferred from the donor parent.After the initial cross, individuals possessing the phenotype of thedonor parent are selected and repeatedly crossed (backcrossed) to therecurrent parent. The resulting parent is expected to have theattributes of the recurrent parent (e.g., cultivar) and the desirabletrait transferred from the donor parent.

The DNA molecules of the present invention can be used as molecularmarkers in a marker assisted breeding (MAB) method. DNA molecules of thepresent invention can be used in methods (such as, AFLP markers, RFLPmarkers, RAPD markers, SNPs, and SSRs) that identify genetically linkedagronomically useful traits, as is known in the art. Theherbicide-resistance trait can be tracked in the progeny of a cross witha soybean plant of the subject invention (or progeny thereof and anyother soybean cultivar or variety) using the MAB methods. The DNAmolecules are markers for this trait, and MAB methods that are wellknown in the art can be used to track the herbicide-resistance trait(s)in soybean plants where at least one soybean line of the subjectinvention, or progeny thereof, was a parent or ancestor. The methods ofthe present invention can be used to identify any soybean variety havingthe subject event.

Methods of the subject invention include a method of producing aherbicide-tolerant soybean plant wherein said method comprisesintrogessing Event pDAB8264.44.06.1 into a soybean cultivar. Morespecifically, methods of the present invention can comprise crossing twoplants of the subject invention, or one plant of the subject inventionand any other plant. Preferred methods further comprise selectingprogeny of said cross by analyzing said progeny for an event detectableaccording to the subject invention. For example, the subject inventioncan be used to track the subject event through breeding cycles withplants comprising other desirable traits, such as agronomic traits suchas those tested herein in various Examples. Plants comprising thesubject event and the desired trait can be detected, identified,selected, and quickly used in further rounds of breeding, for example.The subject event/trait can also be combined through breeding, andtracked according to the subject invention, with an insect resistanttrait(s) and/or with further herbicide tolerance traits. One embodimentof the latter is a plant comprising the subject event combined with agene encoding resistance to the herbicide dicamba.

Thus, the subject invention can be combined with, for example,additional traits encoding glyphosate resistance (e.g., resistant plantor bacterial glyphosate oxidase (GOX)), glyphosate acetyl transferase(GAT), additional traits for glufosinate resistance (e.g. bialaphosresistance (bar)), traits conferring acetolactate synthase(ALS)-inhibiting herbicide resistance (e.g., imidazolinones [such asimazethapyr], sulfonylureas, triazolopyrimidine sulfonanilide,pyrmidinylthiobenzoates, and other chemistries [Csr1, SurA, et al.]),bromoxynil resistance traits (e.g., Bxn), traits for resistance todicamba herbicide (see, e.g., U.S. 2003/0135879), traits for resistanceto inhibitors of HPPD (4-hydroxlphenyl-pyruvate-dioxygenase) enzyme,traits for resistance to inhibitors of phytoene desaturase (PDS), traitsfor resistance to photosystem II inhibiting herbicides (e.g., psbA),traits for resistance to photosystem I inhibiting herbicides, traits forresistance to protoporphyrinogen oxidase IX (PPO)-inhibiting herbicides(e.g., PPO-1), and traits for resistance to phenylurea herbicides (e.g.,CYP76B1). One or more of such traits can be combined with the subjectinvention to provide the ability to effectively control, delay and/orprevent weed shifts and/or resistance to herbicides of multiple classes.

It will be appreciated by those of skill in the art that the aad-12 geneused in the subject invention also provides resistance to compounds thatare converted to phenoxyacetate auxin herbicides (e.g., 2,4-DB, MCPB,etc.). The butyric acid moiety present in the 2,4-DB herbicide isconverted through β-oxidation to the phytotoxic2,4-dichlorophenoxyacetic acid. Likewise, MCPB is converted throughβ-oxidation to the phytotoxic MCPA. The butanoic acid herbicides arethemselves nonherbicidal, but are converted to their respective acidfrom by β-oxidation within susceptible plants to produce the acetic acidform of the herbicide that is phytotoxic. Plants incapable of rapidβ-oxidation are not harmed by the butanoic acid herbicides. However,plants that are capable of rapid β-oxidation and can convert thebutanoic acid herbicide to the acetic form are subsequently protected byAAD-12.

Methods of applying herbicides are well known in the art. Suchapplications can include tank mixes of more than one herbicide.Preferred herbicides for use according to the subject invention arecombinations of glyphosate, glufosinate, and a phenoxy auxin herbicide(such as 2,4-D; 2,4-DB; MCPA; MCPB). Other preferred combinations induceglyphosate plus 2,4-D or glufosinate plus 2,4-D mixtures. These threetypes of herbicides can be used in advantageous combinations that wouldbe apparent to one skilled in the art having the benefit of the subjectdisclosure. One or more of the subject herbicides can be applied to afield/area prior to planting it with seeds of the subject invention.Such applications can be within 14 days, for example, of planting seedsof the subject invention. One or more of the subject herbicides can alsobe applied after planting prior to emergence. One or more of the subjectherbicides can also be applied to the ground (for controlling weeds) orover the top of the weeds and/or over the top of transgenic plants ofthe subject invention. The subject three herbicides can be rotated orused in combination to, for example, control or prevent weeds that mightto tolerant to one herbicide but not another. Various application timesfor the subject three types of herbicides can be used in various ways aswould be known in the art.

Additionally, the subject event can be stacked with one or moreadditional herbicide tolerance traits, one or more additional input(e.g., insect resistance (e.g., the 812 Event or the 814 Event), fungalresistance, or stress tolerance, et al.) or output (e.g., increasedyield, improved oil profile, improved fiber quality, et al.) traits,both transgenic and nontransgenic. Thus, the subject invention can beused to provide a complete agronomic package of improved crop qualitywith the ability to flexibly and cost effectively control any number ofagronomic pests. Methods to integrate a polynucleotide sequence within aspecific chromosomal site of a plant cell via homologous recombinationhave been described within the art. For instance, site specificintegration as described in U.S. Patent Application Publication No.2009/0111188 A1, describes the use of recombinases or integrases tomediate the introduction of a donor polynucleotide sequence into achromosomal target. In addition, International Patent Application No. WO2008/021207 describes zinc finger mediated-homologous recombination tointegrate one or more donor polynucleotide sequences within specificlocations of the genome. The use of recombinases such as FLP/FRT asdescribed in U.S. Pat. No. 6,720,475, or CRE/LOX as described in U.S.Pat. No. 5,658,772, can be utilized to integrate a polynucleotidesequence into a specific chromosomal site. Finally the use ofmeganucleases for targeting donor polynucleotides into a specificchromosomal location was described in Puchta et al., PNAS USA 93 (1996)pp. 5055-5060).

Other various methods for site specific integration within plant cellsare generally known and applicable (Kumar et al., Trends in Plant Sci.6(4) (2001) pp. 155-159). Furthermore, site-specific recombinationsystems which have been identified in several prokaryotic and lowereukaryotic organisms may be applied to use in plants. Examples of suchsystems include, but are not limited too; the R/RS recombinase systemfrom the pSR1 plasmid of the yeast Zygosaccharomyces rouxii (Araki etal. (1985) J. Mol. Biol. 182: 191-203), and the Gin/gix system of phageMu (Maeser and Kahlmann (1991) Mol. Gen. Genet. 230: 170-176).

In some embodiments of the present invention, it can be desirable tointegrate or stack a new transgene(s) in proximity to an existingtransgenic event. The transgenic event can be considered a preferredgenomic locus which was selected based on unique characteristics such assingle insertion site, normal Mendelian segregation and stableexpression, and a superior combination of efficacy, including herbicidetolerance and agronomic performance in and across multiple environmentallocations. The newly integrated transgenes should maintain the transgeneexpression characteristics of the existing transformants. Moreover, thedevelopment of assays for the detection and confirmation of the newlyintegrated event would be overcome as the genomic flanking sequences andchromosomal location of the newly integrated event are alreadyidentified. Finally, the integration of a new transgene into a specificchromosomal location which is linked to an existing transgene wouldexpedite the introgression of the transgenes into other geneticbackgrounds by sexual out-crossing using conventional breeding methods.

In some embodiments of the present invention, it can be desirable toexcise polynucleotide sequences from a transgenic event. For instancetransgene excision as described in U.S. patent application Ser. No.13/011,666, describes the use of zinc finger nucleases to remove apolynucleotide sequence, consisting of a gene expression cassette, froma chromosomally integrated transgenic event. The polynucleotide sequencewhich is removed can be a selectable marker. Upon excision and removalof a polynucleotide sequence the modified transgenic event can beretargeted by the insertion of a polynucleotide sequence. The excisionof a polynucleotide sequence and subsequent retargeting of the modifiedtransgenic event provides advantages such as re-use of a selectablemarker or the ability to overcome unintended changes to the planttranscriptome which results from the expression of specific genes.

The subject invention discloses herein a specific site on chromosome 6in the soybean genome that is excellent for insertion of heterologousnucleic acids. Also disclosed is a 5′ flanking sequence and a 3′flanking sequence, which can also be useful in identifying and/ortargeting the location of the insertion/targeting site on chromosome 6.Thus, the subject invention provides methods to introduce heterologousnucleic acids of interest into this pre-established target site or inthe vicinity of this target site. The subject invention also encompassesa soybean seed and/or a soybean plant comprising any heterologousnucleotide sequence inserted at the disclosed target site or in thegeneral vicinity of such site, One option to accomplish such targetedintegration is to excise and/or substitute a different insert in placeof the pat expression cassette exemplified herein. In this generalregard, targeted homologous recombination, for example and withoutlimitation, can be used according to the subject invention.

As used herein gene, event or trait “stacking” is combining desiredtraits into one transgenic line, Plant breeders stack transgenic traitsby making crosses between parents that each have a desired trait andthen identifying offspring that have both of these desired traits.Another way to stack genes is by transferring two or more genes into thecell nucleus of a plant at the same time during transformation. Anotherway to stack genes is by re-transforming a transgenic plant with anothergene of interest. For example, gene stacking can be used to combine twoor more different traits, including for example, two or more differentinsect traits, insect resistance trait(s) and disease resistancetrait(s), two or more herbicide resistance traits, and/or insectresistance trait(s) and herbicide resistant trait(s). The use of aselectable marker in addition to a gene of interest can also beconsidered gene stacking.

“Homologous recombination” refers to a reaction between any pair ofnucleotide sequences having corresponding sites containing a similarnucleotide sequence through which the two nucleotide sequences caninteract (recombine) to form a new, recombinant DNA sequence. The sitesof similar nucleotide sequence are each referred to herein as a“homology sequence.” Generally, the frequency of homologousrecombination increases as the length of the homology sequenceincreases. Thus, while homologous recombination can occur between twonucleotide sequences that are less than identical, the recombinationfrequency (or efficiency) declines as the divergence between the twosequences increases. Recombination may be accomplished using onehomology sequence on each of the donor and target molecules, therebygenerating a “single-crossover” recombination product. Alternatively,two homology sequences may be placed on each of the target and donornucleotide sequences. Recombination between two homology sequences onthe donor with two homology sequences on the target generates a“double-crossover” recombination product. If the homology sequences onthe donor molecule flank a sequence that is to be manipulated (e.g., asequence of interest), the double-crossover recombination with thetarget molecule will result in a recombination product wherein thesequence of interest replaces a DNA sequence that was originally betweenthe homology sequences on the target molecule. The exchange of DNAsequence between the target and donor through a double-crossoverrecombination event is termed “sequence replacement.”

The subject event enables transgenic expression of three differentherbicide tolerance proteins resulting in tolerance to combinations ofherbicides that would control nearly all broadleaf and grass weeds. Thismulti-herbicide tolerance trait expression cassette/transgenic insertcan be stacked with other herbicide tolerance traits (e.g., glyphosateresistance, glufosinate resistance, imidazolinone resistance, dicambaresistance, HPPD resistance, bromoxynil resistance, et al.), and insectresistance traits (such as Cry1F, Cry1Ab, Cry1Ac, Cry 34/45, Cry1Be,Cry1Ca, Cry1Da, Cry1Ea, Cry1Fa, vegetative insecticidal proteins(“VIPS”)—including VIP3A, and the like), for example. Additionally, theherbicide tolerance proteins in the expression cassette/transgenicinsert of the subject invention can serve as one or more selectablemarker sto aid in selection of primary transformants of plantsgenetically engineered with a second gene or group of genes.

These combinations of traits give rise to novel methods of controllingweeds (and like) species, due to the newly acquired resistance orinherent tolerance to herbicides (e.g., glyphosate). Thus, novel methodsfor controlling weeds using Event pDAB8264.44.06.1 are within the scopeof the invention.

The use of the subject transgenic traits, stacked or transformedindividually into crops, provides a tool for controlling other herbicidetolerant volunteer crops that do not contain genes for conferringtolerance to phenoxy, pyridyloxy, glyphosate and/or glufosinateherbicides.

A preferred plant, or a seed, of the subject invention comprises in itsgenome the insert sequences, as identified herein, together with atleast 20-500 or more contiguous flanking nucleotides on both sides ofthe insert, as described herein. Unless indicated otherwise, referenceto flanking sequences refers to those identified with respect to SEQ IDNO:1 and SEQ ID NO:2. Again, the subject events include heterologous DNAinserted between the subject flanking genomic sequences immediatelyadjacent to the inserted DNA. All or part of these flanking sequencescould be expected to be transferred to progeny that receives theinserted DNA as a result of a sexual cross of a parental line thatincludes the event.

The subject invention includes tissue cultures of regenerable cells of aplant of the subject invention. Also included is a plant regeneratedfrom such tissue culture, particularly where said plant is capable ofexpressing all the morphological and physiological properties of anexemplified variety. Preferred plants of the subject invention have allthe physiological and morphological characteristics of a plant grownfrom the deposited seed. This invention further comprises progeny ofsuch seed and seed possessing the quality traits of interest.

Manipulations (such as mutation, further transfection, and furtherbreeding) of plants or seeds, or parts thereof, may lead to the creationof what may be termed “essentially derived” varieties. The InternationalUnion for the Protection of New Varieties of Plants (UPOV) has providedthe following guideline for determining if a variety has beenessentially derived from a protected variety:

[A] variety shall be deemed to be essentially derived from anothervariety (“the initial variety”) when

(i) it is predominantly derived from the initial variety, or from avariety that is itself predominantly derived from the initial variety,while retaining the expression of the essential characteristics thatresult from the genotype or combination of genotypes of the initialvariety;

(ii) it is clearly distinguishable from the initial variety; and

(iii) except for the differences which result from the act ofderivation, it conforms to the initial variety in the expression of theessential characteristics that result from the genotype or combinationof genotypes of the initial variety.

UPOV, Sixth Meeting with International Organizations, Geneva, Oct. 30,1992; document prepared by the Office of the Union.

As used herein, a “line” is a group of plants that display little or nogenetic variation between individuals for at least one trait. Such linesmay be created by several generations of self-pollination and selection,or vegetative propagation from a single parent using tissue or cellculture techniques.

As used herein, the terms “cultivar” and “variety” are synonymous andrefer to a line which is used for commercial production.

“Stability” or “stable” means that with respect to the given component,the component is maintained from generation to generation and,preferably, at least three generations at substantially the same level,e.g., preferably ±15%, more preferably ±10%, most preferably ±5%. Thestability may be affected by temperature, location, stress and the timeof planting. Comparison of subsequent generations under field conditionsshould produce the component in a similar manner.

“Commercial Utility” is defined as having good plant vigor and highfertility, such that the crop can be produced by farmers usingconventional farming equipment, and the oil with the describedcomponents can be extracted from the seed using conventional crushingand extraction equipment. To be commercially useful, the yield, asmeasured by seed weight, oil content, and total oil produced per acre,is within 15% of the average yield of an otherwise comparable commercialcanola variety without the premium value traits grown in the sameregion.

“Agronomically elite” means that a line has desirable agronomiccharacteristics such as yield, maturity, disease resistance, and thelike, in addition to the herbicide tolerance due to the subjectevent(s). Agronomic traits, taken individually or in any combination, asset forth in Examples, below, in a plant comprising an event of thesubject invention, are within the scope of the subject invention. Anyand all of these agronomic characteristics and data points can be usedto identify such plants, either as a point or at either end or both endsof a range of chracteristics used to define such plants.

As one skilled in the art will recognize in light of this disclosure,preferred embodiments of detection kits, for example, can include probesand/or primers directed to and/or comprising “junction sequences” or“transition sequences” (where the soybean genomic flanking sequencemeets the insert sequence). For example, this includes a polynucleotideprobes, primers, and/or amplicons designed to identify one or bothjunction sequences (where the insert meets the flanking sequence), asindicated in the Table 1. One common design is to have one primer thathybridizes in the flanking region, and one primer that hybridizes in theinsert. Such primers are often each about at least 15 residues inlength. With this arrangement, the primers can be used togenerate/amplify a detectable amplicon that indicates the presence of anevent of the subject invention. These primers can be used to generate anamplicon that spans (and includes) a junction sequence as indicatedabove.

The primer(s) “touching down” in the flanking sequence is typically notdesigned to hybridize beyond about 200 bases or so beyond the junction.Thus, typical flanking primers would be designed to comprise at least 15residues of either strand within 200 bases into the flanking sequencesfrom the beginning of the insert. That is, primers comprising a sequenceof an appropriate size from (or hybridizing to) residues within 100 to200-500 or so bases from either or both junction sequences identifiedabove are within the scope of the subject invention. Insert primers canlikewise be designed anywhere on the insert, but residues on the insert(including the complement) within 100 to 200-500 or so bases in from thejunction sequence(s) identified above, can be used, for example,non-exclusively for such primer design.

One skilled in the art will also recognize that primers and probes canbe designed to hybridize, under a range of standard hybridization and/orPCR conditions, to segments of sequences exemplified herein (orcomplements thereof), wherein the primer or probe is not perfectlycomplementary to the exemplified sequence. That is, some degree ofmismatch can be tolerated. For an approximately 20 nucleotide primer,for example, typically one or two or so nucleotides do not need to bindwith the opposite strand if the mismatched base is internal or on theend of the primer that is opposite the amplicon. Various appropriatehybridization conditions are provided below. Synthetic nucleotideanalogs, such as inosine, can also be used in probes. Peptide nucleicacid (PNA) probes, as well as DNA and RNA probes, can also be used. Whatis important is that such probes and primers are diagnostic for (able touniquely identify and distinguish) the presence of an event of thesubject invention.

It should be noted that errors in PCR amplification can occur whichmight result in minor sequencing errors, for example. That is, unlessotherwise indicated, the sequences listed herein were determined bygenerating long amplicons from soybean genomic DNAs, and then cloningand sequencing the amplicons. It is not unusual to find slightdifferences and minor discrepancies in sequences generated anddetermined in this manner, given the many rounds of amplification thatare necessary to generate enough amplicon for sequencing from genomicDNAs. One skilled in the art should recognize and be put on notice thatany adjustments needed due to these types of common sequencing errors ordiscrepancies are within the scope of the subject invention.

It should also be noted that it is not uncommon for some genomicsequence to be deleted, for example, when a sequence is inserted duringthe creation of an event. Thus, some differences can also appear betweenthe subject flanking sequences and genomic sequences listed in GENBANK,for example.

Components of the “insert” are illustrated in the Figures and arediscussed in more detail below in the Examples. The DNA polynucleotidesequences of these components, or fragments thereof, can be used as DNAprimers or probes in the methods of the present invention.

In some embodiments of the invention, compositions and methods areprovided for detecting the presence of the transgene/genomic insertionregion, in plants and seeds and the like, from a soybean plant. DNAsequences are provided that comprise the subject transgene/genomicinsertion region junction sequence provided herein, segments comprisinga junction sequence identified herein, and complements of any suchexemplified sequences and any segments thereof. The insertion regionjunction sequence spans the junction between heterologous DNA insertedinto the genome and the DNA from the soybean cell flanking the insertionsite. Such sequences can be diagnostic for the given event.

Based on these insert and border sequences, event-specific primers canbe generated. PCR analysis demonstrated that soybean lines of thesubject invention can be identified in different soybean genotypes byanalysis of the PCR amplicons generated with these event-specific primersets. These and other related procedures can be used to uniquelyidentify these soybean lines. Thus, PCR amplicons derived from suchprimer pairs are unique and can be used to identify these soybean lines.

In some embodiments, DNA sequences that comprise a contiguous fragmentof the novel transgene/genomic insertion region are an aspect of thisinvention. Included are DNA sequences that comprise a sufficient lengthof polynucleotides of transgene insert sequence and a sufficient lengthof polynucleotides of soybean genomic sequence from one or more of theaforementioned soybean plants and/or sequences that are useful as primersequences for the production of an amplicon product diagnostic for oneor more of these soybean plants.

Related embodiments pertain to DNA sequences that comprise at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, or more contiguous nucleotides of a transgene portion of aDNA sequence identified herein, or complements thereof, and a similarlength of flanking soybean DNA sequence (such as SEQ ID NO:1 and SEQ IDNO:2 and segments thereof) from these sequences, or complements thereof.Such sequences are useful as DNA primers in DNA amplification methods.The amplicons produced using these primers are diagnostic for any of thesoybean events referred to herein. Therefore, the invention alsoincludes the amplicons produced by such DNA primers and homologousprimers.

This invention also includes methods of detecting the presence of DNA,in a sample, that corresponds to the soybean event referred to herein.Such methods can comprise: (a) contacting the sample comprising DNA witha primer set that, when used in a nucleic acid amplification reactionwith DNA from at least one of these soybean events, produces an ampliconthat is diagnostic for said event(s); (b) performing a nucleic acidamplification reaction, thereby producing the amplicon; and (c)detecting the amplicon.

Further detection methods of the subject invention include a method ofdetecting the presence of a DNA, in a sample, corresponding to saidevent, wherein said method comprises: (a) contacting the samplecomprising DNA with a probe that hybridizes under stringenthybridization conditions with DNA from at least one of said soybeanevents and which does not hybridize under the stringent hybridizationconditions with a control soybean plant (non-event-of-interest DNA); (b)subjecting the sample and probe to stringent hybridization conditions;and (c) detecting hybridization of the probe to the DNA.

In still further embodiments, the subject invention includes methods ofproducing a soybean plant comprising Event pDAB8264.44.06.1, whereinsaid method comprises the steps of: (a) sexually crossing a firstparental soybean line (comprising an expression cassettes of the presentinvention, which confers said herbicide resistance trait to plants ofsaid line) and a second parental soybean line (that lacks this herbicidetolerance trait) thereby producing a plurality of progeny plants; and(b) selecting a progeny plant by the use of molecular markers. Suchmethods may optionally comprise the further step of back-crossing theprogeny plant to the second parental soybean line to producing atrue-breeding soybean plant that comprises said herbicide tolerancetrait.

According to another aspect of the invention, methods of determining thezygosity of progeny of a cross with said event is provided. Said methodscan comprise contacting a sample, comprising soybean DNA, with a primerset of the subject invention. Said primers, when used in a nucleic-acidamplification reaction with genomic DNA from at least one of saidsoybean events, produces a first amplicon that is diagnostic for atleast one of said soybean events. Such methods further compriseperforming a nucleic acid amplification reaction, thereby producing thefirst amplicon; detecting the first amplicon; and contacting the samplecomprising soybean DNA with said primer set (said primer set, when usedin a nucleic-acid amplification reaction with genomic DNA from soybeanplants, produces a second amplicon comprising the native soybean genomicDNA homologous to the soybean genomic region; and performing a nucleicacid amplification reaction, thereby producing the second amplicon. Themethods further comprise detecting the second amplicon, and comparingthe first and second amplicons in a sample, wherein the presence of bothamplicons indicates that the sample is heterozygous for the transgeneinsertion.

DNA detection kits can be developed using the compositions disclosedherein and methods well known in the art of DNA detection. The kits areuseful for identification of the subject soybean event DNA in a sampleand can be applied to methods for breeding soybean plants containingthis DNA. The kits contain DNA sequences homologous or complementary tothe amplicons, for example, disclosed herein, or to DNA sequenceshomologous or complementary to DNA contained in the transgene geneticelements of the subject events. These DNA sequences can be used in DNAamplification reactions or as probes in a DNA hybridization method. Thekits may also contain the reagents and materials necessary for theperformance of the detection method.

A “probe” is an isolated nucleic acid molecule to which is attached aconventional detectable label or reporter molecule (such as aradioactive isotope, ligand, chemiluminescent agent, or enzyme). Such aprobe is complementary to a strand of a target nucleic acid, in the caseof the present invention, to a strand of genomic DNA from one of saidsoybean events, whether from a soybean plant or from a sample thatincludes DNA from the event. Probes according to the present inventioninclude not only deoxyribonucleic or ribonucleic acids but alsopolyamides and other probe materials that bind specifically to a targetDNA sequence and can be used to detect the presence of that target DNAsequence. An “isolated” polynucleotide connotes that the polynucleotideis in a non-natural state—operably linked to a heterologous promoter,for example. A “purified” protein likewise connotes that the protein isin a non-natural state.

“Primers” are isolated/synthesized nucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, then extended alongthe target DNA strand by a polymerase, e.g., a DNA polymerase. Primerpairs of the present invention refer to their use for amplification of atarget nucleic acid sequence, e.g., by the polymerase chain reaction(PCR) or other conventional nucleic-acid amplification methods.

Probes and primers are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496,497, 498, 499, or 500 polynucleotides or more in length. Such probes andprimers hybridize specifically to a target sequence under highstringency hybridization conditions. Preferably, probes and primersaccording to the present invention have complete sequence similaritywith the target sequence, although probes differing from the targetsequence and that retain the ability to hybridize to target sequencesmay be designed by conventional methods.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989. PCR-primer pairs can be derived from a knownsequence, for example, by using computer programs intended for thatpurpose.

Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm (and, if necessary, to correct)the disclosed sequences by conventional methods, e.g., by re-cloning andsequencing such sequences.

The nucleic acid probes and primers of the present invention hybridizeunder stringent conditions to a target DNA sequence. Any conventionalnucleic acid hybridization or amplification method can be used toidentify the presence of DNA from a transgenic event in a sample.Nucleic acid molecules or fragments thereof are capable of specificallyhybridizing to other nucleic acid molecules under certain circumstances.As used herein, two nucleic acid molecules are said to be capable ofspecifically hybridizing to one another if the two molecules are capableof forming an anti-parallel, double-stranded nucleic acid structure. Anucleic acid molecule is said to be the “complement” of another nucleicacid molecule if they exhibit complete complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the molecules is complementary to a nucleotide ofthe other. Two molecules are said to be “minimally complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under at least conventional“low-stringency” conditions. Similarly, the molecules are said to be“complementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another underconventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., 1989. Departures fromcomplete complementarity are therefore permissible, as long as suchdepartures do not completely preclude the capacity of the molecules toform a double-stranded structure. In order for a nucleic acid moleculeto serve as a primer or probe it need only be sufficiently complementaryin sequence to be able to form a stable double-stranded structure underthe particular solvent and salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. The term “stringent conditions” is functionallydefined with regard to the hybridization of a nucleic-acid probe to atarget nucleic acid (i.e., to a particular nucleic-acid sequence ofinterest) by the specific hybridization procedure discussed in Sambrooket al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52and 9.56-9.58. Accordingly, the nucleotide sequences of the inventionmay be used for their ability to selectively form duplex molecules withcomplementary stretches of DNA fragments.

Depending on the application envisioned, one can use varying conditionsof hybridization to achieve varying degrees of selectivity of probetowards target sequence. For applications requiring high selectivity,one will typically employ relatively stringent conditions to form thehybrids, e.g., with regards to endpoint TaqMan and real-time PCRapplications, one will select 1.5 mM to about 4.0 mM MgCl2 attemperature of about 60° C. to about 75° C. and may vary hold times, asdescribed herein, for increasing stringency. For other hybridizationtechniques one will typically employ relatively low salt and/or hightemperature conditions, such as provided by about 0.02 M to about 0.15 MNaCl at temperatures of about 50° C. to about 70° C. Stringentconditions, for example, could involve washing the hybridization filterat least twice with high-stringency wash buffer (0.2×SSC, 0.1% SDS, 65°C.). Appropriate stringency conditions which promote DNA hybridization,for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C. are known to those skilled inthe art. For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a highstringency of about 0.2×SSC at 50° C. In addition, the temperature inthe wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged. Such selective conditions tolerate little, if any, mismatchbetween the probe and the template or target strand. Detection of DNAsequences via hybridization is well-known to those of skill in the art,and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 areexemplary of the methods of hybridization analyses.

In a particularly preferred embodiment, a nucleic acid of the presentinvention will specifically hybridize to one or more of the primers (oramplicons or other sequences) exemplified or suggested herein, includingcomplements and fragments thereof, under high stringency conditions. Inone aspect of the present invention, a marker nucleic acid molecule ofthe present invention has the nucleic acid sequence as set forth hereinin one of the exemplified sequences, or complements and/or fragmentsthereof.

In another aspect of the present invention, a marker nucleic acidmolecule of the present invention shares between 80% and 100% or 90% and100% sequence identity with such nucleic acid sequences. In a furtheraspect of the present invention, a marker nucleic acid molecule of thepresent invention shares between 95% and 100% sequence identity withsuch sequence. Such sequences may be used as markers in plant breedingmethods to identify the progeny of genetic crosses. The hybridization ofthe probe to the target DNA molecule can be detected by any number ofmethods known to those skilled in the art, these can include, but arenot limited to, fluorescent tags, radioactive tags, antibody based tags,and chemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic-acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofnucleic-acid amplification of a target nucleic acid sequence that ispart of a nucleic acid template. For example, to determine whether thesoybean plant resulting from a sexual cross contains transgenic eventgenomic DNA from the soybean plant of the present invention, DNAextracted from a soybean plant tissue sample may be subjected to nucleicacid amplification method using a primer pair that includes a primerderived from flanking sequence in the genome of the plant adjacent tothe insertion site of inserted heterologous DNA, and a second primerderived from the inserted heterologous DNA to produce an amplicon thatis diagnostic for the presence of the event DNA. The amplicon is of alength and has a sequence that is also diagnostic for the event. Theamplicon may range in length from the combined length of the primerpairs plus one nucleotide base pair, and/or the combined length of theprimer pairs plus about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286,287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342,343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356,357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384,385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468,469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496,497, 498, 499, or 500, 750, 1000, 1250, 1500, 1750, 2000, or morenucleotide base pairs (plus or minus any of the increments listedabove). Alternatively, a primer pair can be derived from flankingsequence on both sides of the inserted DNA so as to produce an ampliconthat includes the entire insert nucleotide sequence. A member of aprimer pair derived from the plant genomic sequence may be located adistance from the inserted DNA sequence. This distance can range fromone nucleotide base pair up to about twenty thousand nucleotide basepairs. The use of the term “amplicon” specifically excludes primerdimers that may be formed in the DNA thermal amplification reaction.

Nucleic-acid amplification can be accomplished by any of the variousnucleic-acid amplification methods known in the art, including thepolymerase chain reaction (PCR). A variety of amplification methods areknown in the art and are described, inter alia, in U.S. Pat. No.4,683,195 and U.S. Pat. No. 4,683,202. PCR amplification methods havebeen developed to amplify up to 22 kb of genomic DNA. These methods aswell as other methods known in the art of DNA amplification may be usedin the practice of the present invention. The sequence of theheterologous transgene DNA insert or flanking genomic sequence from asubject soybean event can be verified (and corrected if necessary) byamplifying such sequences from the event using primers derived from thesequences provided herein followed by standard DNA sequencing of the PCRamplicon or of the cloned DNA.

The amplicon produced by these methods may be detected by a plurality oftechniques. Agarose gel electrophoresis and staining with ethidiumbromide is a common well known method of detecting DNA amplicons.Another such method is Genetic Bit Analysis where an DNA oligonucleotideis designed which overlaps both the adjacent flanking genomic DNAsequence and the inserted DNA sequence. The oligonucleotide isimmobilized in wells of a microwell plate. Following PCR of the regionof interest (using one primer in the inserted sequence and one in theadjacent flanking genomic sequence), a single-stranded PCR product canbe hybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labeledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotideis designed that overlaps the adjacent genomic DNA and insert DNAjunction. The oligonucleotide is hybridized to single-stranded PCRproduct from the region of interest (one primer in the inserted sequenceand one in the flanking genomic sequence) and incubated in the presenceof a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. DNTPs are added individually and theincorporation results in a light signal that is measured. A light signalindicates the presence of the transgene insert/flanking sequence due tosuccessful amplification, hybridization, and single or multi-baseextension.

Fluorescence Polarization is another method that can be used to detectan amplicon of the present invention. Following this method, anoligonucleotide is designed which overlaps the genomic flanking andinserted DNA junction. The oligonucleotide is hybridized tosingle-stranded PCR product from the region of interest (one primer inthe inserted DNA and one in the flanking genomic DNA sequence) andincubated in the presence of a DNA polymerase and a fluorescent-labeledddNTP. Single base extension results in incorporation of the ddNTP.Incorporation can be measured as a change in polarization using afluorometer. A change in polarization indicates the presence of thetransgene insert/flanking sequence due to successful amplification,hybridization, and single base extension.

TAQMAN (PE Applied Biosystems, Foster City, Calif.) is a method ofdetecting and quantifying the presence of a DNA sequence. Briefly, aFRET oligonucleotide probe is designed that overlaps the genomicflanking and insert DNA junction. The FRET probe and PCR primers (oneprimer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. During specific amplification, Taq DNA polymerase cleans andreleases the fluorescent moiety away from the quenching moiety on theFRET probe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection.Briefly, a FRET oligonucleotide probe is designed that overlaps theflanking genomic and insert DNA junction. The unique structure of theFRET probe results in it containing secondary structure that keeps thefluorescent and quenching moieties in close proximity. The FRET probeand PCR primers (one primer in the insert DNA sequence and one in theflanking genomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal results. Afluorescent signal indicates the presence of the flankinggenomic/transgene insert sequence due to successful amplification andhybridization.

Having disclosed a location in the soybean genome that is excellent foran insertion, the subject invention also includes a soybean seed and/ora soybean plant comprising at least one non-aad12/pat/2mepsps codingsequence in or around the general vicinity of this genomic location. Oneoption is to substitute a different insert in place of the insertexemplified herein. In these general regards, targeted homologousrecombination, for example, can be used according to the subjectinvention. This type of technology is the subject of, for example, WO03/080809 A2 and the corresponding published U.S. application (U.S.2003/0232410). Thus, the subject invention includes plants and plantcells comprising a heterologous insert (in place of or with multi-copiesof the exemplified insert), flanked by all or a recognizable part of theflanking sequences identified herein as SEQ ID NO:1 and SEQ ID NO:2. Anadditional copy (or additional copies) of the exemplified insert or anyof its components could also be targeted for insertion in this/thesemanner(s).

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

The following examples are included to illustrate procedures forpracticing the invention and to demonstrate certain preferredembodiments of the invention. These examples should not be construed aslimiting. It should be appreciated by those of skill in the art that thetechniques disclosed in the following examples represent specificapproaches used to illustrate preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in these specific embodimentswhile still obtaining like or similar results without departing from thespirit and scope of the invention. Unless otherwise indicated, allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

The following abbreviations are used unless otherwise indicated.

-   -   bp base pair    -   ° C. degrees Celcius    -   DNA deoxyribonucleic acid    -   DIG digoxigenin    -   EDTA ethylenediaminetetraacetic acid    -   kb kilobase    -   μg microgram    -   μL microliter    -   mL milliliter    -   M molar mass    -   OLP overlapping probe    -   PCR polymerase chain reaction    -   PTU plant transcription unit    -   SDS sodium dodecyl sulfate    -   SOP standard operating procedure    -   SSC a buffer solution containing a mixture of sodium chloride        and sodium citrate, pH 7.0    -   TBE a buffer solution containing a mixture of Tris base, boric        acid and EDTA, pH 8.3    -   V volts

Examples Example 1: Transformation and Selection of the 2mEPSPS andAAD-12 Soybean Event 8264.44.06.1

Transgenic soybean (Glycine max) containing the Soybean Event8264.44.06.1 was generated through Agrobacterium-mediated transformationof soybean cotyledonary node explants. The disarmed Agrobacterium strainEHA101 (Hood et al., 2006), carrying the binary vector pDAB8264 (FIG. 1)containing the selectable marker, pat, and the genes of interest, aad-12and 2mepsps v1, within the T-strand DNA region, was used to initiatetransformation.

Agrobacterium-mediated transformation was carried out using a modifiedprocedure of Zeng et al. (2004). Briefly, soybean seeds (cv Maverick)were germinated on basal media and cotyledonary nodes were isolated andinfected with Agrobacterium. Shoot initiation, shoot elongation, androoting media were supplemented with cefotaxime, timentin and vancomycinfor removal of Agrobacterium. Glufosinate selection was employed toinhibit the growth of non-transformed shoots. Selected shoots weretransferred to rooting medium for root development and then transferredto soil mix for acclimatization of plantlets.

Terminal leaflets of selected plantlets were leaf painted withglufosinate to screen for putative transformants. The screened plantletswere transferred to the greenhouse, allowed to acclimate and thenleaf-painted with glufosinate to reconfirm tolerance and deemed to beputative transformants. The screened plants were sampled and molecularanalyses for the confirmation of the selectable marker gene and/or thegene of interest were carried out. T₀ plants were allowed to selffertilize in the greenhouse to give rise to T₁ seed.

This event, Soybean Event 8264.44.06.1, was generated from anindependent transformed isolate. The T₁ plants were backcrossed andintrogressed into elite varieties over subsequent generations. The eventwas selected based on its unique characteristics such as singleinsertion site, normal Mendelian segregation and stable expression, anda superior combination of efficacy, including herbicide tolerance andagronomic performance. The following examples contain the data whichwere used to characterize Soybean Event 8264.44.06.1.

Example 2: Characterization of AAD-12, 2mEPSPS and PAT Protein inSoybean Event 8264.44.06.1

The biochemical properties of the recombinant AAD-12, 2mEPSPS and PATprotein derived from the transgenic soybean event pDAB8264.44.06.1 werecharacterized. Quantitative enzyme-linked immunosorbent assay (ELISA)was used to characterize the biochemical properties of the protein andconfirm expression of AAD-12, PAT and 2mEPSPS protein.

Example 2.1: Expression of the AAD-12 Protein in Plant Tissues

Levels of AAD-12 protein were determined in soybean event 8264.44.06.1.The soluble, extractable AAD-12 protein was measured using aquantitative enzyme-linked immunosorbent assay (ELISA) method fromsoybean leaf tissue.

Samples of soybean tissues were isolated from the test plants andprepared for expression analysis. The AAD-12 protein was extracted fromsoybean plant tissues with a phosphate buffered saline solutioncontaining the detergent Tween-20 (PBST) containing 0.5% Bovine SerumAlbumin (BSA). The plant tissue was centrifuged; the aqueous supernatantwas collected, diluted with appropriate buffer as necessary, andanalyzed using an AAD-12 ELISA kit in a sandwich format. The kit wasused following the manufacturer's suggested protocol.

Detection analysis was performed to investigate the expression stabilityand heritability both vertically (between generations) and horizontally(between lineages of the same generation) in soybean event 8264.44.06.1.At the T4 generation soybean event 8264.44.06.1 expression was stable(not segregating) and consistent across all lineages. Field expressionlevel studies were performed on soybean event; average expression acrossall lineages was approximately 200-400 ng/cm².

Example 2.2: Expression of the 2mEPSPS Protein in Plant Tissues

Levels of 2mEPSPS protein were determined in soybean event 8264.44.06.1.The soluble, extractable 2mEPSPS protein was measured using aquantitative enzyme-linked immunosorbent assay (ELISA) method fromsoybean leaf tissue.

Samples of soybean tissues were isolated from the test plants andprepared for expression analysis. The 2mEPSPS protein was extracted fromsoybean plant tissues with a phosphate buffered saline solutioncontaining the detergent Tween-20 (PBST) containing 0.5% Bovine SerumAlbumin (BSA). The plant tissue was centrifuged; the aqueous supernatantwas collected, diluted with appropriate buffer as necessary, andanalyzed using a 2mEPSPS ELISA kit in a sandwich format. The kit wasused following the manufacturer's suggested protocol.

Detection analysis was performed to investigate the expression stabilityand heritability both vertically (between generations) and horizontally(between lineages of the same generation) in soybean event 8264.44.06.1.At the T4 generation soybean event 8264.44.06.1 expression was stable(not segregating) and consistent across all lineages. Field expressionlevel studies were performed on soybean event 8264.44.06.1. Averageexpression across all lineages was approximately 5,000-17,500 ng/cm².These expression levels were higher than the positive control whichexpressed the 2mEPSPS protein.

Example 2.3: Expression of the PAT Protein in Plant Tissues

Levels of PAT protein were determined in soybean event 8264.44.06.1. Thesoluble, extractable PAT protein was measured using a quantitativeenzyme-linked immunosorbent assay (ELISA) method from soybean leaftissue.

Samples of soybean tissues were isolated from the test plants andprepared for expression analysis. The PAT protein was extracted fromsoybean plant tissues with a phosphate buffered saline solutioncontaining the detergent Tween-20 (PBST) containing 0.5% Bovine SerumAlbumin (BSA). The plant tissue was centrifuged; the aqueous supernatantwas collected, diluted with appropriate buffer as necessary, andanalyzed using a PAT ELISA kit in a sandwich format. The kit was usedfollowing the manufacturer's suggested protocol.

Detection analysis was performed to investigate the expression stabilityand heritability both vertically (between generations) and horizontally(between lineages of the same generation) in soybean event 8264.44.06.1.At the T4 generation soybean event 8264.44.06.1 expression was stable(not segregating) and consistent across all lineages. Field expressionlevel studies were performed on soybean event 8264.44.06.1. Averageexpression across all lineages was approximately 15-25 ng/cm².

Example 3: Cloning and Characterization of DNA Sequence in the Insertand the Flanking Border Regions of Soybean Event pDAB8264.44.06.1

To characterize and describe the genomic insertion site, the sequence ofthe flanking genomic T-DNA border regions of soybean eventpDAB8264.44.06.1 were determined. In total, 2,578 bp of soybean eventpDAB8264.44.06.1 genomic sequence was confirmed, comprising 570 bp of 5′flanking border sequence (SEQ ID NO:1), 1,499 bp of 3′ flanking bordersequence (SEQ ID NO:2). PCR amplification based on the soybean EventpDAB8264.44.06.1 border sequences validated that the border regions wereof soybean origin and that the junction regions are unique sequences forevent pDAB8264.44.06.1. The junction regions could be used forevent-specific identification of soybean event pDAB8264.44.06.1. Inaddition, the T-strand insertion site was characterized by amplifying agenomic fragment corresponding to the region of the identified flankingborder sequences from the genome of wild type soybean. Comparison ofsoybean event pDAB8264.44.06.1 with the wild type genomic sequencerevealed about 4,357 bp deletion from the original locus. Overall, thecharacterization of the insert and border sequence of soybean eventpDAB8264.44.06.1 indicated that an intact copy of the T-strand waspresent in the soybean genome.

TABLE 2 Primers and sequences used to analyze Soybean Event pDAB8264.44.06.1SEQ ID Primer Size NO: Name (bp) Sequence (5′ to 3′) Purpose SEQ ID4406_ 25 AGGTTGTCATTCCGCTGAAGAAGAT confirmation of 5′ border NO: 3 WF1genomic DNA, used with ED_v1_C1 SEQ ID 4406_ 25CACAGTGGACAATTCTGATTTCTGG confirmation of 5′ border NO: 4 WF2genomic DNA, used with ED_v1_C SEQ ID 4406_ 25 GGATTGCATCTGAAACGGATCATATconfirmation of 5′ border NO: 5 WF3 genomic DNA, used with ED_v1_C1SEQ ID 4406_ 25 GGAATGTTGAACCACCCATGATTAA confirmation of 5′ borderNO: 6 WF4 genomic DNA, used with ED_v1_C SEQ ID 4406- 25CATGTATGTTGTTGTCGTTGCCTTG confirmation of 3′ border NO: 7 WR5genomic DNA, used with PAT_12 SEQ ID 4406- 25 AACATTTTGAAATCGGTTCCAAGGAconfirmation of 3′ border NO: 8 WR6 genomic DNA, used with PAT_12 SEQ ID4406- 25 AGGCTCAGGCCAACAACATTAATTT confirmation of 3′ border NO: 9 WR7genomic DNA, used with PAT_12 SEQ ID 4406- 27GGAGAGAAGTCGCAACAGTGATTACAT confirmation of 3′ border NO: 10 WR8genomic DNA, used with PAT_12 SEQ ID ED_v1_ 26GAGTAAAGGAGACCGAGAGGATGGTT confirmation of 5′ border NO: 11 C1genomic DNA, used with 4406_WF1, 4406_WF2, 4406_WF3, or 4406_WF4, SEQ IDPAT_12 24 GAACGCTTACGATTGGACAGTTGA confirmation of 3′ border NO: 12genomic DNA, used with 4406_WR5, 4406_WR62, 4406_WR7, or 4406_WR8

TABLE 3 PCR conditions for amplification of border regions andevent-specific sequences in soybean event pDAB8264.44.06.1. Pre- FinalTarget PCR denature Denature Anneal Extension Extension Sequence PrimerSet Mixture (° C./min) (° C./sec.) (° C./sec.) (° C./min:sec) (° C./min)5′ border 4406- D 95/3 98/10 66/30 68/4:00 72/10 WF1/ED_v1_C1 32 cycles5′ border 4406- D 95/3 98/10 66/30 68/4:00 72/10 WF3/ED_v1_C1 32 cycles3′ border 4406- D 95/3 98/10 66/30 68/4:00 72/10 WR5/PAT_12 35 cycles 3′border 4406- D 95/3 98/10 66/30 68/4:00 72/10 WR7/PAT_12 32 cycles 3′border 4406- D 95/3 98/10 66/30 68/4:00 72/10 WR8/PAT_12 35 cyclesAcross the 4406-WF1/4406- D 95/3 98/10 66/30  68/10:00 72/10 insert WR532 cycles locus Across the 4406-WF3/4406- D 95/3 98/10 66/30  68/10:0072/10 insert WR7 32 cycles locus

TABLE 4 PCR mixture for amplification of border regions and eventspecific sequences in soybean event pDAB8264.44.06.1. 1 x reactionReagent (μL) PCR Mixture A H20 0.8 AccPrime pfx SuperMix 20 — — — — 10uM primer 0.2 gDNA digestion 1 — — rxn vol: 22 PCR Mixture B H20 14.610X LA Taq buffer 2 MgCl2 (25 mM) 0.6 dNTP (2.5 uM) 1.6 10 uM primer 0.1gDNA digestion 1 LA Taq (5 U/ul) 0.1 rxn vol: 20 PCR Mixture C H20 2810X PCR buffer II (Mg-plus) 5 MgCl₂[25 mM] 1.5 dNTP[2.5 mM] 8 AdaptorPCR primer (10 μM) 1 GOI nested primer (10 μM) 1 DNA binded Beads 5 LATaq (5 U/ul) 0.5 rxn vol: 50 PCR Mixture D H20 11.6 10X PCR buffer II(Mg-plus) 2 MgCl₂[25 mM] 0.6 dNTP[2.5 mM] 3.2 primer1 (10 μM) 0.4primer2 (10 μM) 0.4 DNA Template 0.2 LA Taq (5 U/ul) 1.6 rxn vol: 20

Example 3.1: Confirmation of Soybean Genomic Sequences

The 5′ and 3′ flanking borders aligned to a Glycine max whole genomeshotgun sequence from chromosome 6, indicating that the transgene ofsoybean event pDAB8264.44.06.1 was inserted in soybean genome chromosome6. To confirm the insertion site of soybean event pDAB8264.44.06.1transgene from the soybean genome, PCR was carried out with differentpairs of primers (FIG. 2 and Table 3). Genomic DNA from soybean eventpDAB8264.44.06.1 and other transgenic or non-transgenic soybean lineswas used as a template. Thus, to confirm if the 5′ border sequences arecorrect, 2mepsps specific primers, for example ED_v1_C1 (SEQ ID NO:11),and two primers designed according to the cloned 5′ end border sequenceand/or its alignment sequence on soybean genome chromosome 6, designated4406-WF1 (SEQ ID NO:3) and 4406-WF3 (SEQ ID NO:5), were used foramplifying the DNA segment that spans the 2mepsps gene to 5′ end bordersequence. Similarly, for confirmation of the cloned 3′ end bordersequence, a pat specific primer, for example PAT-12 (SEQ ID NO:12), andthree primers designed according to the cloned 3′ end border sequence,designated 4406-WR5 (SEQ ID NO:7), 4406-WR7 (SEQ ID NO:9) and 4406-WR8(SEQ ID NO:10), were used for amplifying DNA segments that span the patgene to 3′ end border sequence. DNA fragments with predicted sizes wereamplified only from the genomic DNA of soybean event pDAB8264.44.06.1with each primer pair, one primer located on the flanking border ofsoybean event pDAB8264.44.06.1 and one transgene specific primer, butnot from DNA samples from other transgenic soybean lines ornon-transgenic control. The results indicate that the cloned 5′ and 3′border sequences are the flanking border sequences of the T-strandinsert for soybean event pDAB8264.44.06.1.

To further confirm the DNA insertion in the soybean genome, a PCRamplification spanning the two soybean sequences was completed. Twoprimers designed according to the 5′ end border sequence, 4406-WF1 (SEQID NO:3) and 4406-WF3 (SEQ ID NO:5), and two primers for the 3′ endborder sequence, 4406-WR5 (SEQ ID NO:7) and 4406-WR7 (SEQ ID NO:9), wereused to amplify DNA segments which contained the entire transgene, the5′ end border sequence, and the 3′ border sequence. As expected, PCRamplification with the primer pair of 4406-WF1 (SEQ ID NO:3) and4406-WR5 (SEQ ID NO:7) amplified an approximately 12 kb DNA fragmentfrom the genomic DNA of soybean event pDAB8264.44.06.1 and a 6 kb DNAfragment from the non-transgenic soybean controls and other soybeantransgenic lines. Similarly, PCR reactions completed with the primerpair of 4406-WF3 (SEQ ID NO:5) and 4406-WR7 (SEQ ID NO:9) produced anapproximately 12 kb DNA fragment from the sample of soybean eventpDAB8264.44.06.1 and a 6 kb DNA fragment from all the other soybeancontrol lines, correspondingly. These results demonstrated that thetransgene of soybean event pDAB8264.44.06.1 was inserted into the siteof soybean genome chromosome 6. Aligning the identified 5′ and 3′ bordersequences of soybean event pDAB8264.44.06.1 with a Glycine max wholegenome shotgun sequence from chromosome 6 revealed about 4.4 kb deletionfrom the original locus. (FIG. 3).

Example 4: Soybean Event pDAB8264.44.06.1 Characterization Via SouthernBlot

Southern blot analysis was used to establish the integration pattern ofsoybean event pDAB8264.44.06.1. These experiments generated data whichdemonstrated the integration and integrity of the aad-12, pat and2mepsps v1 transgenes within the soybean genome. Soybean eventpDAB8264.44.06.1 was characterized as a full length, simple integrationevent containing a single copy of the aad-12, pat and 2mepsps v1 PTUfrom plasmid pDAB8264.

Southern blot data suggested that a T-strand fragment inserted into thegenome of soybean event pDAB8264.44.06.1. Detailed Southern blotanalysis was conducted using a probe specific to the aad-12, pat and2mepsps v1 insert, contained in the T-strand integration region ofpDAB8264, and descriptive restriction enzymes that have cleavage siteslocated within the plasmid and produce hybridizing fragments internal tothe plasmid or fragments that span the junction of the plasmid withsoybean genomic DNA (border fragments). The molecular weights indicatedfrom the Southern hybridization for the combination of the restrictionenzyme and the probe were unique for the event, and established itsidentification patterns. These analyses also showed that the plasmidfragment had been inserted into soybean genomic DNA withoutrearrangements of the aad-12, pat and 2mepsps v1 PTU.

Example 4.1: Soybean Leaf Sample Collection and Genomic DNA (gDNA)Isolation

Genomic DNA was extracted from leaf tissue harvested from individualsoybean plants containing soybean event pDAB8264.44.06.1. In addition,gDNA was isolated from a conventional soybean plant, Maverick, whichcontains the genetic background that is representative of the substanceline, absent the aad-12 and 2mepsps v1 genes. Individual genomic DNA wasextracted from lyophilized leaf tissue following the standardcetyltrimethylammonium bromide CTAB method. Following extraction, theDNA was quantified spectrofluorometrically using Pico Green reagent(Invitrogen, Carlsbad, Calif.). The DNA was then visualized on anagarose gel to confirm values from the Pico Green analysis and todetermine the DNA quality.

Example 4.2: DNA Digestion and Separation

For Southern blot molecular characterization of soybean eventpDAB8264.44.06.1, ten micrograms (10 μg) of genomic DNA was digested.Genomic DNA from the soybean pDAB8264.44.06.1 and non-transgenic soybeanline Maverick was digested by adding approximately five units ofselected restriction enzyme per μg of DNA and the corresponding reactionbuffer to each DNA sample. Each sample was incubated at approximately37° C. overnight. The restriction enzymes BstZ17I, HinDIII, NcoI, NsiI,and Pad were used individually for the digests (New England Biolabs,Ipswich, Mass.). In addition, a positive hybridization control samplewas prepared by combining plasmid DNA, pDAB8264 with genomic DNA fromthe non-transgenic soybean variety, Maverick. The plasmid DNA/genomicDNA cocktail was digested using the same procedures and restrictionenzyme as the test samples. After the digestions were incubatedovernight, NaCl was added to a final concentration of 0.1M and thedigested DNA samples were precipitated with isopropanol. Theprecipitated DNA pellet was resuspended in 20 μl of 1× loading buffer(0.01% bromophenol blue, 10.0 mM EDTA, 5.0% glycerol, 1.0 mM Tris pH7.5). The DNA samples and molecular size markers were thenelectrophoresed through 0.85% agarose gels with 0.4×TAE buffer (FisherScientific, Pittsburgh, Pa.) at 35 volts for approximately 18-22 hoursto achieve fragment separation. The gels were stained with ethidiumbromide (Invitrogen, Carlsbad, Calif.) and the DNA was visualized underultraviolet (UV) light

Example 4.3: Southern Transfer and Membrane Treatment

Southern blot analysis was performed essentially as described by,Memelink, J.; Swords, K.; Harry J.; Hoge, C.; (1994) Southern, Northern,and Western Blot Analysis. Plant Mol. Biol. Manual F1:1-23. Briefly,following electrophoretic separation and visualization of the DNAfragments, the gels were depurinated with 0.25M HCl for approximately 20minutes, and then exposed to a denaturing solution (0.4 M NaOH, 1.5 MNaCl) for approximately 30 minutes followed by neutralizing solution(1.5 M NaCl, 0.5 M Tris pH 7.5) for at least 30 minutes. Southerntransfer was performed overnight onto nylon membranes using a wickingsystem with 10×SSC. After transfer the DNA was bound to the membrane byUV crosslinking following by briefly washing membrane with a 2×SSCsolution. This process produced Southern blot membranes ready forhybridization.

Example 4.4: DNA Probe Labeling and Hybridization

The DNA fragments bound to the nylon membrane were detected using alabeled probe. Probes were generated by a PCR-based incorporation of adigoxigenin (DIG) labeled nucleotide, [DIG-11]-dUTP, into the DNAfragment amplified from plasmid pDAB8264 using primers specific to geneelements. Generation of DNA probes by PCR synthesis was carried outusing a PCR DIG Probe Synthesis Kit (Roche Diagnostics, Indianapolis,Ind.) following the manufacturer's recommended procedures.

Labeled probes were analyzed by agarose gel electrophoresis to determinetheir quality and quantity. A desired amount of labeled probe was thenused for hybridization to the target DNA on the nylon membranes fordetection of the specific fragments using the procedures essentially asdescribed for DIG Easy Hyb Solution (Roche Diagnostics, Indianapolis,Ind.). Briefly, nylon membrane blots containing fixed DNA were brieflywashed with 2×SSC and pre-hybridized with 20-25 mL of pre-warmed DIGEasy Hyb solution in hybridization bottles at approximately 45-55° C.for about 2 hours in a hybridization oven. The pre-hybridizationsolution was then decanted and replaced with approximately 15 mL ofpre-warmed DIG Easy Hyb solution containing a desired amount of specificprobes denatured by boiling in a water bath for approximately fiveminutes. The hybridization step was then conducted at approximately45-55° C. overnight in the hybridization oven.

At the end of the probe hybridization, DIG Easy Hyb solutions containingthe probes were decanted into clean tubes and stored at approximately−20° C. These probes could be reused for twice according to themanufacturer's recommended procedure. The membrane blots were rinsedbriefly and washed twice in clean plastic containers with low stringencywash buffer (2×SSC, 0.1% SDS) for approximately five minutes at roomtemperature, followed by washing twice with high stringency wash buffer(0.1×SSC, 0.1% SDS) for 15 minutes each at approximately 65° C. Themembrane blots briefly washed with 1× Maleic acid buffer from the DIGWash and Block Buffer Set (Roche Diagnostics, Indianapolis, Ind.) forapproximately 5 minutes. This was followed by blocking in a 1× blockingbuffer for 2 hours and an incubation with anti-DIG-AP (alkalinephosphatase) antibody (Roche Diagnostics, Indianapolis, Ind.) in 1×blocking buffer also for a minimum of 30 minutes. After 2-3 washes with1× washing buffer, specific DNA probes remain bound to the membraneblots and DIG-labeled DNA standards were visualized using CDP-StarChemiluminescent Nucleic Acid Detection System (Roche Diagnostics,Indianapolis, Ind.) following the manufacturer's recommendation. Blotswere exposed to chemiluminescent film for one or more time points todetect hybridizing fragments and to visualize molecular size standards.Films were developed with an All-Pro 100 Plus film developer (KonicaMinolta, Osaka, Japan) and images were scanned. The number and sizes ofdetected bands were documented for each probe (Table 5). DIG-labeled DNAMolecular Weight Marker II (DIG MWM II) and DIG-labeled DNA MolecularWeight Marker VII (DIG MWM VII), visible after DIG detection asdescribed, were used to determine hybridizing fragment size on theSouthern blots.

TABLE 5 Length of probes used in Southern analysis of soybean eventpDAB8264.44.06.1. Length Probe Name Genetic Element (bp) 2mEPSPS 2mEPSPS1238 aad-12 aad-12 671 specR Spectinomycin resistance gene 750 OriRepOri Rep 852 trfA Replication initiation protein trfA 1119

Example 4.5: Southern Blot Results

Expected and observed fragment sizes with a particular digest and probe,based on the known restriction enzyme sites of the aad-12 and 2mepspsPTU, are given in Table 6. Expected fragment sizes are based on theplasmid map of pDAB8264 and observed fragment sizes are approximateresults from these analyses and are based on the indicated sizes of theDIG-labeled DNA Molecular Weight Marker II and Mark VII fragments.

Two types of fragments were identified from these digests andhybridizations: internal fragments where known enzyme sites flank theprobe region and are completely contained within the insertion region ofthe aad-12 and 2mepsps PTU PTU, and border fragments where a knownenzyme site is located at one end of the probe region and a second siteis expected in the soybean genome. Border fragment sizes vary by eventbecause, in most cases, DNA fragment integration sites are unique foreach event. The border fragments provide a means to locate a restrictionenzyme site relative to the integrated DNA and to evaluate the number ofDNA insertions. Southern blot analyses completed on multiple generationsof soybean containing event pDAB8264.44.06.1 produced data whichsuggested that a low copy, intact aad-12 and 2mepsps PTU from plasmidpDAB8264 was inserted into the soybean genome of soybean eventpDAB8264.44.06.1.

TABLE 6 Predicted and Observed Hybridizing Fragments in Southern BlotAnalysis. Expected Observed Fragment Fragment DNA Restriction Sizes SizeProbe Enzymes Samples (bp) ¹ (bp)² aad-12 BstZ17I pDAB8264 4994 ~5000Maverick none none Soybean Event 4994 ~5000 pDAB8264.44.06.1 Hind IIIpDAB8264 4731 ~4700 Maverick none none Soybean Event >4078   ~7400pDAB8264.44.06.1 Nco I pDAB8264 7429 ~7400 Maverick none none SoybeanEvent >3690   ~3800 pDAB8264.44.06.1 Nsi I pDAB8264 4974 ~5000 Mavericknone none Soybean Event 4974 ~5000 pDAB8264.44.06.1 Pac I pDAB8264 6768~6800 Maverick none none Soybean Event 6768 ~6800 pDAB8264.44.06.12mEPSPS BstZ17I pDAB8264 11024  ~11000  Maverick none none SoybeanEvent >4858   ~16000  pDAB8264.44.06.1 Nco I pDAB8264 5203 ~5200Maverick none none Soybean Event >3756   ~6100 pDAB8264.44.06.1 Nsi IpDAB8264 11044  11000 Maverick none Soybean Event >5199   ~5300pDAB8264.44.06.1 Pac I pDAB8264 6768 ~6800 Maverick none none SoybeanEvent 6768 ~6800 pDAB8264.44.06.1 SpecR Hind III pDAB8264 9322 ~9300Maverick none none Soybean Event none none pDAB8264.44.06.1 OriRep + PacI pDAB8264 9210 ~9200 trfA Maverick none none Soybean Event none nonepDAB8264.44.06.1

The restriction enzymes NcoI and HinD III bind and cleave uniquerestriction sites in plasmid pDAB8264. Subsequently, these enzymes wereselected to characterize the aad-12 gene insert in soybean eventpDAB8264.44.06.1. Border fragments of greater than 4,078 bp or greaterthan 3,690 bp were predicted to hybridize with the probe following HinDIII and NcoI digests, respectively (Table 6). Single aad-12hybridization bands of approximately 7,400 bp and approximately 3,800 bpwere observed when HinDIII and NcoI were used, respectively. Thehybridization of the probe to bands of this size suggests the presenceof a single site of insertion for the aad-12 gene in the soybean genomeof soybean event pDAB8264.44.06.1. Restriction enzymes BstZ17I, NsiI andPad was selected to release a fragment which contains the aad-12 planttranscription unit (PTU; promoter/gene/terminator) (Table 6). Thepredicted approximately 5,000, approximately 5,000, and approximately6,800 bp fragments were observed with the probe following BstZ17I, NsiIand Pad digestions, respectively. Results obtained with the enzymedigestion of the pDAB8264.44.06.1 samples followed by probehybridization indicated that an intact aad-12 PTU from plasmid pDAB8264was inserted into the soybean genome of soybean event pDAB8264.44.06.1.In addition, the molecular weight sizes of the hybridization bandsproduced for the HinDIII, NcoI, NsiI, and BstZ17I restriction fragmentsindicate that the aad-12 PTU also contained the linked pat PTU.

The restriction enzymes BstZ17I, NcoI and NsiI bind and cleaverestriction sites in plasmid pDAB8264. Subsequently, these enzymes wereselected to characterize the 2mepsps gene insert in soybean eventpDAB8264.44.06.1. Border fragments of greater than 4,858 bp, greaterthan 3,756, or greater than 5,199 bp were predicted to hybridize withthe probe following the BstZ17I, NcoI and NsiI digests respectively(Table 6). Single 2mepsps hybridization bands of approximately 16,000bp, approximately 6,100 bp and approximately 5,300 bp were observed whenBstZ17I, NcoI and NsiI were used, respectively. The hybridization of theprobe to bands of this size suggests the presence of a single site ofinsertion for the 2mepsps gene in the soybean genome of soybean eventpDAB8264.44.06.1. Restriction enzyme Pad was selected to release afragment which contains the 2mepsps plant transcription unit (PTU;promoter/gene/terminator) (Table 6). The predicted approximately 6,800bp fragment was observed with the probe following the Pad digestions.Results obtained with the enzyme digestion of the pDAB8264.44.06.1sample followed by probe hybridization indicated that an intact 2mepspsPTU from plasmid pDAB8264 was inserted into the soybean genome ofsoybean event pDAB8264.44.06.1.

Example 4.6: Absence of Backbone Sequences

Southern blot analysis was also conducted to verify the absence of thespectinomycin resistance gene (specR), Ori Rep element and replicationinitiation protein trfA (trf A element) in soybean eventpDAB8264.44.06.1. No specific hybridization to spectinomycin resistance,Ori Rep element or trf A element is expected when appropriate positive(pDAB8264 plus Maverick) and negative (Maverick) controls are includedfor Southern analysis. Following Hind III digestion and hybridizationwith specR specific probe, one expected size band of approximately 9,300bp was observed in the positive control sample (pDAB8264 plus maverick)but absent from samples of the negative control and soybean eventpDAB8264.44.06.1. Similarly, one expected size band of approximately9,200 bp was detected in the positive control sample (pDAB8264 plusmaverick) but absent from the samples of the negative control andsoybean event pDAB8264.44.06.1 after Pac I digestion and hybridizationwith mixture of OriRep specific probe and trfA specific probe. Thesedata indicate the absence of spectinomycin resistance gene, Ori Repelement and replication initiation protein trfA in soybean eventpDAB8264.44.06.1.

Example 5: Agronomic, Yield and Herbicide Tolerance Evaluation

The agronomic characteristics and herbicide tolerance of soybean EventpDAB8264.44.06.1 were studied in yield trials at multiple geographicallocales during a single growing season. No agronomically meaningfulunintended differences were observed between soybean EventpDAB8264.44.06.1 and the Maverick control plants. The results of thestudy demonstrated that soybean Event pDAB8264.44.06.1 was agronomicallyequivalent to the Maverick control plants. In addition, soybean EventpDAB8264.44.06.1 provided robust herbicide tolerance when sprayed with atankmix of glyphosate and 2,4-D.

The following agronomic characteristics were measured and recorded forall test entries at each location.

-   -   1.) Emergence: Calculated by dividing Stand count by number of        seeds planted in a one meter section and multiplying by 100.    -   2.) Seedling Vigor at V1: Vigor is an overall estimate of the        health of the plot. Results were rated on a scale of 0-100% with        0% representing a plot with all dead plants and 100%        representing plots that look very healthy.    -   3.) Rated overall visual crop injury, chlorosis and necrosis at        1 day, 7 days, and 14 days after V3 chemical application.        Observations were made for any signs of epinasty which is        typical of 2,4-D injury. Epinasty is exhibited as twisting or        drooping of leaves and stems. All crop injuries used a 0 to 100%        scale, where 0% indicates no injury and 100% indicates complete        plant death.    -   4.) Flowering date: This measurement records the date when 50%        of the plants in the plot begin to flower. The number of days        from planting to when 50% of the plants in each plot were        flowering was recorded.    -   5.) Stand count at R2 or R1: Is a visual estimate of the average        vigor of plants in each plot, determined by counting the number        of plants in a representative one meter section of one row per        plot, and taking note at the R2 or R1 growth stage.    -   6.) Rated overall visual crop injury, chlorosis and necrosis at        1 day, 7 days, and 14 days after R2 chemical application.        Observations were made for any signs of epinasty which is        typical of 2,4-D injury. Epinasty is exhibited as twisting or        drooping of leaves and stems. All crop injuries used a 0 to 100%        scale where 0% indicates no injury and 100% indicates complete        plant death.    -   7.) Disease incidence at R6 growth stage: Is a visual estimate        of disease incidence used to record the severity of disease in        the plot. Rated on a scale of 0-100%. Where 0% indicates no        disease present and 100% indicates all plants in plot had        disease.    -   8.) Insect damage at R6 growth stage: Is a visual estimate of        insect damage used to record the severity of insect damage in        the plot. Recorded the percentage of plant tissue in the plot        damaged by insects using a 0-100% scale. Where 0% indicates no        insect damage present and 100% indicates all plants in plot had        insect damage.    -   9.) Plant height at senescence: The average height of the plants        in each plot was recorded. Plants were measured from the soil        surface to the tip after the leaves had fallen. Measurements        were recorded in centimeters. If the plot was lodged, a        representative group of plants were stood-up to obtain a        measurement.    -   10.) Days to maturity. Recorded date when 95% of the pods in a        plot reached physiological maturity and the plants were a dry        down color. The numbers of days to elapse since planting were        calculated.    -   11.) Lodging: Recorded a visual estimate of lodging severity at        harvest time. Recorded on a 0 to 100% scale, where 0% indicates        no lodging and 100% indicates all plants in a plot flat on the        ground.    -   12.) Shattering: Recorded a visual estimate of pod shattering at        harvest time. Recorded as an estimate of percentage of pods        shattered per plot. 0-100% scale with 0% indicating no        shattering and 100% indicating all pods shattered.    -   13.) Yield: Recorded the weight of grain harvested from each        plot. Harvested the entire 2 row plot and recorded seed weight        and moisture. Calculations of bu/acre were made by adjusting to        13% moisture.    -   14.) 100 seed weight: For each plot 100 seeds were counted out        and the weight was recorded in grams.

Herbicide tolerance of soybean Event pDAB8264.44.06.1 was assessedfollowing the application of a tankmix of 2,4-D and glyphosate at 2,185g ae/ha mixed with 2% weight per weight ammonium sulfate (AMS). Theherbicides were sprayed as a V3/R2 sequential herbicide treatment. Thisherbicide treatment was completed by spraying soybean plants at the V3growth stage of development followed by a second sequential applicationat the R2 growth stage of development. The V3 growth stage ischaracterized when the unifoliolate and first three trifoliolate leavesare fully developed. The R2 growth stage is characterized by a singleopen flower at one of the two uppermost nodes on the main stem with afully developed leaf.

These trials were set up using a randomized complete block design withfour replications for every treatment. Each plot was 2 rows wide androws were spaced 30 inches apart. Plots were planted to a total lengthof 12.5 ft with a 2.5 to 3.0 ft alley between plots. Maverick controlplants were expected to die from herbicide applications so they weregrown in a separate plot; away from the transgenic soybean plant rows.

The results of soybean Event pDAB8264.44.06.1 sprayed with the 2,4-D andglyphosate herbicide tank mix as compared to unsprayed soybean EventpDAB8264.44.06.1 are summarized. Table 7 presents the means from ananalysis comparing soybean Event pDAB8264.44.06.1 sprayed with a tankmixof 2,4-D and glyphosate to unsprayed soybean Event pDAB8264.44.06.1. Theherbicide application did not damage soybean Event pDAB8264.44.06.1,these plants performed equivalently as compared to unsprayed soybeanEvent pDAB8264.44.06.1 plants for the reported agronomic characteristicslisted in Table 7. With the exception of some early transient injury 1and 7 daa (days after application) at the V3 stage of development and at1, 7 and 14 daa at the R2 stage of development, soybean EventpDAB8264.44.06.1 showed robust tolerance to the 2,4-D and glyphosatetank mix. In contrast, none of the Maverick plants were surviving afterbeing sprayed with the herbicide treatment.

TABLE 7 Comparison of soybean Event pDAB8264.44.06.1 sprayed andunsprayed with a tank mix of 2,4-D glyphosate. soybean EventpDAB8264.44.06.1 Trait: Agronomic Characteristics Sprayed Non-sprayedEmergence (%) 90.2 84.0 Vigor V1-V3 (%) 93.4 88.4 Rated overall visualcrop injury 1.3 0.0 after V3 herbicide application; Injury 1 daa (%)Rated overall visual crop injury 1.1 0.0 after V3 herbicide application;Injury 7 daa (%) Rated overall visual crop injury 0.0 0.0 after V3herbicide application; 14 daa (%) Days to flower (days from planting)38.6 38.5 Stand count R2 26.1 22.5 Rated overall visual crop injury 2.80.4 after R2 herbicide application; Injury 1 daa (%) Rated overallvisual crop injury 2.8 0.0 after R2 herbicide application; Injury 7 daa(%) Rated overall visual crop injury 1.7 0.1 after R2 herbicideapplication; Injury 14 daa (%) Disease incidence (%) 1.5 1.2 Insectdamage (%) 6.9 7.6 Height (cm) 112.3 110.3 Maturity (days from planting)114.0 113.7 Lodging (%) 16.4 18.1 Shattering (%) 0.1 0.1 Yield (bu/acre)44.8 43.9 100 seed weight (g) 12.3 12.1

Agronomic equivalence of soybean Event pDAB8264.44.06.1 as compared tothe control line, Maverick, was assessed. These trials were set up usinga block design with two replications. Each plot was 2 rows wide and rowswere spaced 30 inches apart. Plots were planted to a total length of12.5 ft with a 2.5 to 3.0 foot alley between plots.

Table 8 presents the means from the analysis comparing the agronomicequivalence of soybean Event pDAB8264.44.06.1 with the control line,Maverick. The agronomic data is indicative that soybean EventpDAB8264.44.06.1 performs equivalently to Maverick plants, and does notresult in agronomically meaningful unintended differences.

TABLE 8 Comparison of soybean Event pDAB8264.44.06.1to Maverick controllines in yield trials. Maverick pDAB8264.44.06.1 Emergence (%) 86.2 A83.2 A Vigor V1 (1 poor-9 good) 91.0 A 89.7 A Days to flower (days from41.2 A 40.7 A planting) Stand count R1 22.7 A 22.2 A Disease incidence(%) 1.8 A 2.1 A Insect damage (%) 7.8 A 8.0 A Height (cm) 110.3 A 112.3A Maturity (days from planting) 119.7 A 119.1 A Lodging (%) 16.1 B 20.6A Shattering 0.2 A 0.4 A Yield (bu/acre) 45.7 A 43.7 A 100 seed weight13.2 A 12.6 B For each trait values not followed by the same letter aredifferent according to Student's T-distribution statistical analysis.

Example 6: Event Specific TaqMan Assay

Two event specific TAQMAN assays were developed to detect the presenceof soybean event pDAB8264.44.06.1 and to determine zygosity status ofplants in breeding populations. Soybean event pDAB8264.44.06.1 containsthe T-strand of the binary vector pDAB8264 (FIG. 1). For specificdetection of soybean event pDAB8264.44.06.1, specific Taqman primers andprobes were designed according to the DNA sequences located in the 5′(SEQ ID NO:14) or 3′ (SEQ ID NO:15) insert-to-plant junction (FIG. 4).One event specific assay for soybean event pDAB8264.44.06.1 was designedto specifically detect a 98 bp DNA fragment (SEQ ID NO:16) that spansthe 5′ integration junction using two primers and a target-specific MGBprobe synthesized by Applied Biosystems (ABI) containing the FAMreporter at its 5′end. Another event specific assay for soybean eventpDAB8264.44.06.1 was designed to specifically target a 131 bp DNAfragment (SEQ ID NO:17) that spans the 3′ integration junction using twospecific primers and a target-specific MGB probe synthesized by ABIcontaining the FAM reporter at its 5′end. Specificity of this Taqmandetection method for soybean event pDAB8264.44.06.1 was tested against11 different events which contain the 2mEPSPS and aad-12 PTUs and acontrol non-transgenic soybean variety (Maverick) in duplex format withthe soybean specific endogenous reference gene, GMFL01-25-J19 (Glycinemax cDNA, GenBank: AK286292.1).

Example 6.1: gDNA Isolation

gDNA samples of 11 different soybean events and non-transgenic soybeanvarieties were tested in this study. Genomic DNA was extracted usingmodified Qiagen MagAttract plant DNA kit (Qiagen, Valencia, Calif.).Fresh soybean leaf discs, 8 per sample, were used for gDNA extraction.The gDNA was quantified with the Pico Green method according to vendor'sinstructions (Molecular Probes, Eugene, Oreg.). Samples were dilutedwith DNase-free water resulting in a concentration of 10 ng/μL for thepurpose of this study.

Example 6.2: Taqman Assay and Results

Specific Taqman primers and probes were designed for a soybean eventpDAB8264.44.06.1 specific Taqman assay. These reagents can be used withthe conditions listed below to detect the transgene within soybean eventpDAB8264.44.06.1. Table 9 lists the primer and probe sequences that weredeveloped specifically for the detection of soybean eventpDAB8264.44.06.1.

TABLE 9  Taqman PCR Primers and Probes. Event Target Reaction SEQ ID NO:Name Description Sequence SEQ ID 4406_5′F Event specificTTGTTCTTGTTGTTTCCT NO: 18 forward Primer CTTTAGGA SEQ ID 4406_5′REvent specific GACCTCAATTGCGAGCTT NO: 19 reverse Primer TCTAAT SEQ ID4406_5′P Event specific 5′FAM/CATGGAGGTCCG NO: 20 probe used  AATAG-MGBwith 4406_5′F and 4406_5′R SEQ ID 4406_3′F Event specificAAACGTCCGCAATGTGTT NO: 21 forward Primer ATTAAG SEQ ID 4406_3′REvent specific CGTTGCCTTGTTCCACAT NO: 22 reverse Primer ATCA SEQ ID4406_3′P Event specific 5′FAM/ACAGAGAACGAA NO: 23 probe used  TGTC-MGBwith 4406_3′F and 4406_3′R Reference System Reaction SEQ ID NO: NameDescription 5′ to 3′ sequence SEQ ID GMS116 Forward PrimerGTAATATGGGCTCAGAGG NO: 24 F AATGGT SEQ ID GMS116 Reverse PrimerATGGAGAAGAACATTGGA NO: 25 R ATTGC SEQ ID GMS116 Probe 5′HEX/CCATGGCCCGGTNO: 26 Probe ACCATCTGGTC/ 3BHQ_1/3′

The multiplex PCR conditions for amplification are as follows: 1× RochePCR Buffer, 0.4 μM event specific forward primer, 0.4 μM event specificreverse primer, 0.4 μM Primer GMS116 F, 0.4 μM Primer GMS116 R, 0.2 μMEvent specific probe, 0.2 μM GMS116 Probe, 0.1% PVP, 20 ng gDNA in atotal reaction of 10 μl. The cocktail was amplified using the followingconditions: i) 95° C. for 10 min., ii) 95° C. for 10 sec, iii) 60° C.for 30 sec, iv) 72° C. for 1 sec v) repeat step ii-iv for 35 cycles, v)40° C. hold. The Real time PCR was carried out on the Roche LightCycler480. Data analysis was based on measurement of the crossing point (Cpvalue) determined by LightCycler 480 software, which is the PCR cyclenumber when the rate of change in fluorescence reaches its maximum.

The Taqman detection method for soybean event pDAB8264.44.06.1 wastested against 11 different events which contain the 2mEPSPS and aad-12PTUs and non-transgenic soybean varieties in duplex format with soybeanspecific endogenous reference gene, GMFL01-25-J19 (GenBank: AK286292.1).The assays specifically detected the soybean event pDAB8264.44.06.1 anddid not produce or amplify any false-positive results from the controls(i.e. the 11 different events which contain the 2mEPSPS and aad-12 PTUsand non-transgenic soybean varieties). The event specific primers andprobes can be used for the detection of the soybean eventpDAB8264.44.06.1 and these conditions and reagents are applicable forzygosity assays.

Example 7: Full Length Sequence of Soybean Event pDAB8264.44.06.1

SEQ ID NO:27 provides the full length sequence of soybean EventpDAB8264.44.06.1. This sequence contains the 5′ genomic flankingsequence, the integrated T-strand insert from pDAB8264 and the 3′genomic flanking sequence. With respect to SEQ ID NO:27, residues 1-1494are 5′ genomic flanking sequence, residues 1495-1497 are a three basepair insertion, residues 1498-11,774 are the pDAB8264 T-strand insert,and residues 11,775-13,659 are 3′ flanking sequence. The junctionsequence or transition with respect to the 5′ end of the insert thusoccurs at residues 1494-1495 of SEQ ID NO:27. The junction sequence ortransition with respect to the 3′ end of the insert thus occurs atresidues 11,774-11,775 of SEQ ID NO:27. SEQ ID NO:27 is thepolynucleotide sequence of soybean Event pDAB8264.44.06.1 and wasassembled from an alignment of multiple PCR contigs which were producedvia PCR amplification reactions and sequenced using the ABI Big Dye®Terminator sequencing reaction kit (Applied Biosystems, Foster City,Calif.).

Example 8: Breeding Stack of Soybean Event pDAB8264.44.06.1 and SoybeanInsect Tolerant Event pDAB9582.812.9.1 Example 8.1: Sexual Crossing ofSoybean Event pDAB8264.44.06.1 and Soybean Insect Tolerant EventpDAB9582.812.9.1

Soybean event pDAB8264.44.06.1 was sexually crossed with soybean eventpDAB9582.812.9.1. The anthers of soybean event pDAB8264.44.06.1 weremanually rubbed across the stigma of soybean event pDAB9582.812.9.1,thereby fertilizing soybean event pDAB9582.812.9.1. The resulting F1progeny which contained integration events from both soybean eventpDAB9582.812.9.1 and soybean event pDAB8264.44.06.1 were screened fortolerance to 2,4-D and glyphosate herbicides to identify progeny plantswhich contained both integration events. Next, the F1 progeny plantswere self-fertilized to produce an F2 offspring which was confirmed tosegregate independently for both events. The F2 plants were sprayed witha single herbicide application containing both 2,4-D (1120 g ae/ha) andglyphosate (1120 g ae/ha). The resulting F2 plants were screened using aTaqman zygosity based assay to identify plants that were homozygous forboth events. Selfing of these F2 homozygous plants produced an F3offspring that were homozygous for both soybean event pDAB9582.812.9.1and soybean event pDAB8264.44.06.1. The resulting event was labeled assoybean event pDAB9582.812.9.1::pDAB8264.44.06.1.

Example 8.2: Determination of the Zygosity Status of Soybean EventpDAB9582.812.9.1::pDAB8264.44.06.1

To determine the zygosity status of plants produced from the breedingcross of soybean event pDAB8264.44.06.1 and soybean eventpDAB9582.812.9.1, separate event specific TAQMAN® assays were developedto detect the presence of either the pDAB9582.812.9.1 orpDAB8264.44.06.1 integration events. Segregating F2 plants, producedfrom the self fertilization of a breeding cross of soybean eventpDAB9582.812.9.1 and soybean event pDAB8264.44.06.1, were tested withthese event specific TAQMAN® assays to identify individual plants whichcontained both soybean event pDAB9582.812.9.1 and soybean eventpDAB8264.44.06.1, and were homozygous for both events.

gDNA Isolation

gDNA samples from segregating F2 plants of the breeding stack of soybeanevent pDAB9582.812.9.1::pDAB8264.44.06.1 were tested in this study.Fresh soybean leaf discs, 4 per plant, were collected from 3,187segregating F2 plants of the breeding stack of soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1. Genomic DNA was extracted from thesesamples using a modified Qiagen MagAttract Plant DNA Kit® (Qiagen,Valencia, Calif.).

TAQMAN® Assay and Results

TAQMAN® primers and probes as previously described were designed for theuse of individual event specific assays for soybean eventspDAB9582.812.9.1 (U.S. Provisional Application No. 61/471,845) andpDAB8264.44.06.1 (described above). These reagents were used with theconditions listed below to determine the zygosity of each integrationevent contained within the breeding stack of soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1.

The multiplex PCR conditions for amplification are as follows: 1× RochePCR Buffer, 0.4 μM event pDAB8264.44.06.1 specific forward primer, 0.4μM event pDAB8264.44.06.1 specific reverse primer 0.4 μM eventpDAB9582.812.9.1 specific forward primer, 0.4 μM event pDAB9582.812.9.1specific reverse primer, 0.4 μM Primer GMS116 F, 0.4 μM Primer GMS116 R,0.2 μM Event pDAB9582.812.9.1 specific probe, 0.2 μM EventpDAB8264.44.06.1 specific probe, 0.2 μM GMS116 Probe, 0.1% PVP, 20 nggDNA in a total reaction of 10 μl. The cocktail was amplified using thefollowing conditions: i) 95° C. for 10 min., ii) 95° C. for 10 sec, iii)60° C. for 30 sec, iv) 72° C. for 1 sec v) repeat step ii-iv for 35cycles, v) 40° C. hold. The Real time PCR was carried out on the RocheLightCycler® 480. Data analysis was based on measurement of the crossingpoint (Cp value) determined by LightCycler® 480 software, which is thePCR cycle number when the rate of change in fluorescence reaches itsmaximum.

A total of 3,187 segregating F2 plants, produced from the breeding crossof soybean event pDAB9582.812.9.1 and soybean event pDAB8264.44.06.1were tested with the event specific TAQMAN® assays to determine thezygosity of individual plants for both soybean event pDAB9582.812.9.1and soybean event pDAB8264.44.06.1. The results from these assaysindicated that soybean event pDAB9582.812.9.1 and soybean eventpDAB8264.44.06.1 were both present and detected in 2,360 plants. Thezygosity status (also described as ploidy level) of each integrationevent is indicated in Table 9b. Of the 2,360 identified plants, 237 weredetermined to contain two copies of soybean event pDAB9582.812.9.1 andsoybean event pDAB8264.44.06.1.

TABLE 9b Event specific TAQMAN ® zygosity analysis of the breeding stackof soybean event pDAB9582.812.9.1::pDAB8264.44.06.1 Zygosity status forNumber pDAB9582.812.9.1::pDAB8264.44.06.1 of plantsHomozygous::Homozygous 237 Homozygous::Hemizygous 506 Homozygous::Null287 Hemizygous::Homozygous 542 Hemizygous::Hemizygous 1075Hemizygous::Null 540

Example 8.3: Characterization of Protein Expression in the BreedingStack of Soybean Event pDAB9582.812.9.1::pDAB8264.44.06.1

The biochemical properties of the recombinant Cry1F, Cry1Ac, AAD12,2mEPSPS, and PAT proteins expressed in the breeding stack of soybeanevent pDAB9582.812.9.1::pDAB8264.44.06.1 were characterized. An EnzymeLinked Immunosorbent Assay (ELISA) was used to quantify the expressionof PAT. Comparatively, Cry1Ac/Cry1F and AAD12/2mEPSPS proteins werequantified by multiplexed immunoassays utilizing electrochemiluminescenttechnology from Meso-Scale Discovery (MSD, Gaithersburg, Md.).Collectively, these assays were used to characterize the biochemicalproperties and confirm the robust expression of these proteins in thebreeding stack of soybean event pDAB9582.812.9.1::pDAB8264.44.06.1.

Expression of the PAT Protein in Plant Tissues

Levels of PAT protein were determined in the breeding stack of F3soybean event pDAB9582.812.9.1::pDAB8264.44.06.1 which were identifiedto be homozygous for both event pDAB9582.812.9.1 and eventpDAB8264.44.06.1 integrations. The levels of PAT protein expressed fromsoybean event pDAB9582.812.9.1::pDAB8264.44.06.1 was compared to theparental events, soybean event pDAB9582.812.9.1 and soybean eventpDAB8264.44.06.1.

The soluble, extractable PAT protein was obtained from soybean leaftissue and measured using a quantitative ELISA method (APS 014,Envirologix, Portland, Me.). Samples of soybean leaf tissues wereisolated from greenhouse grown test plants at the unifoliate to V1 stageand prepared for expression analysis. The PAT protein was extracted fromsoybean plant tissues with a phosphate buffered saline solutioncontaining the detergent Tween-20 (PBST) and 1% polyvinylpyrrolidone 40(PVP-40). The samples were then extracted using a GenoGrinder® at 1500rpm for 5 minutes. The plant extract was centrifuged; the aqueoussupernatant was collected, diluted with appropriate buffer as necessary,and analyzed using the PAT ELISA kit in a sandwich format. The kit wasused following the manufacturer's suggested protocol (Envirologix,Portland, Me.).

Detection analysis was performed to investigate the expression andheritability of soybean event pDAB9582.812.9.1::pDAB8264.44.06.1. The F3generation of the breeding stack, soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 expressed PAT at higherconcentrations than either the parental events, pDAB9582.812.9.1 andpDAB8264.44.06.1. The increased concentration of PAT in soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 breeding stack was expected. Thehigher concentrations of PAT are a result of soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 containing twice as many copies ofthe pat coding sequence as compared to either of the parental events(Table 10).

TABLE 10 Average PAT protein expression from soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1, and parental events (soybean eventpDAB9582.812.9.1 and soybean event pDAB8264.44.06.1). Average PATExpression Soybean Event (ng/cm²) pDAB9582.812.9.1::pDAB8264.44.06.138.0 pDAB9582.812.9.1 11.0 pDAB8264.44.06.1 13.3

Expression of the Cry1F and Cry1Ac Proteins in Plant Tissues

Levels of Cry1F and Cry1Ac protein were determined in the breeding stackof F3 soybean event pDAB9582.812.9.1::pDAB8264.44.06.1 which wereidentified to be homozygous for both event pDAB9582.812.9.1 and eventpDAB8264.44.06.1 integrations. The levels of Cry1F and Cry1Ac proteinexpressed from soybean event pDAB9582.812.9.1::pDAB8264.44.06.1 wascompared to the parental event, soybean event pDAB9582.812.9.1.

The soluble, extractable Cry1F and Cry1Ac protein was obtained fromsoybean leaf tissue and measured using a multiplexedelectrochemiluminescent MSD assay. Samples of soybean leaf tissue wereisolated from greenhouse grown plants at the unifoliate to V1 stage andprepared for expression analysis. The Cry1F and Cry1Ac protein wasextracted from soybean plant tissues with a phosphate buffered salinesolution containing the detergent Tween-20 (PBST) and 1%polyvinylpyrrolidone 40 (PVP-40). The samples were then extracted usinga GenoGrinder® at 1500 rpm for 5 minutes. The plant extract wascentrifuged; the aqueous supernatant was collected, diluted withappropriate buffer as necessary, and analyzed using a Cry1F/Cry1Acmultiplex MSD assay from Meso-Scale Discovery. The kit was usedfollowing the manufacturer's suggested protocol.

Detection analysis was performed to investigate the expression andheritability of soybean event pDAB9582.812.9.1::pDAB8264.44.06.1. The F3generation of the breeding stack of soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 expressed Cry1F and Cry1Ac proteinsat concentrations higher than the parental soybean eventpDAB9582.812.9.1. (Table 11). These results indicate that soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 plants contained a functionallyexpressing copy of the cry1F and cry1Ac coding sequences which wereinherited from the parental line, soybean event pDAB9582.812.9.1.

TABLE 11 Average Cry1Ac and Cry1F protein expression from soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 as compared to parental eventpDAB9582.812.9.1 Average Average Cry1Ac Cry1F Expression ExpressionSoybean Event (ng/cm²) (ng/cm²) pDAB9582.812.9.1::pDAB8264.44.06.1 27.1140.5 pDAB9582.812.9.1 20.8 112.9Expression of the AAD12 and 2mEPSPS Proteins in Plant Tissues

Levels of AAD12 and 2mEPSPS protein were determined in the breedingstack of F3 soybean event pDAB9582.812.9.1::pDAB8264.44.06.1 which wereidentified to be homozygous for both event pDAB9582.812.9.1 and eventpDAB8264.44.06.1 integrations. The levels of AAD12 and 2mEPSPS proteinexpressed from soybean event pDAB9582.812.9.1::pDAB8264.44.06.1 wascompared to the parental event, Soybean Event pDAB8264.44.06.1.

The soluble, extractable AAD12 and 2mEPSPS protein was obtained fromsoybean leaf tissue and measured using a multiplexedelectrochemiluminescent MSD assay. Samples of soybean leaf tissue wereisolated from greenhouse grown plants at the unifoliate to V1 stage andprepared for expression analysis. The AAD12 and 2mEPSPS protein wasextracted from soybean plant tissues with a phosphate buffered salinesolution containing the detergent Tween-20 (PBST) and 1%polyvinylpyrrolidone 40 (PVP-40). The samples were then extracted usinga GenoGrinder® at 1500 rpm for 5 minutes. The plant extract wascentrifuged; the aqueous supernatant was collected, diluted withappropriate buffer as necessary, and analyzed using a AAD12 and 2mEPSPSmultiplex MSD assay from Meso-Scale Discovery. The kit was usedfollowing the manufacturer's suggested protocol.

Detection analysis was performed to investigate the expression andheritability of soybean event pDAB9582.812.9.1::pDAB8264.44.06.1. The F3generation of the breeding stack of soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 expressed AAD12 and 2mEPSPS proteinsat concentrations higher than the parental soybean eventpDAB8264.44.06.1. (Table 12). These results indicated that soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 plants contained a functionallyexpressing copy of the aad-12 and 2mEPSPS coding sequences which wereinherited from the parental line, soybean event pDAB8264.44.06.1.

TABLE 12 Average AAD12 and 2mEPSPS protein expression from soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 as compared to parental soybean eventpDAB8264.44.06.1 Average Average AAD12 2mEPSPS Expression ExpressionSoybean Event (ng/cm²) (ng/cm²) pDAB9582.812.9.1::pDAB8264.44.06.1 479.7410.3 pDAB8264.44.06.1 320.4 328.9

Example 8.4: Herbicide Tolerance of the Breeding Stack of Soybean EventpDAB9582.812.9.1::pDAB8264.44.06.1

Herbicide tolerance of the breeding stack, soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 was assayed during two growingseasons. Soybean event pDAB9582.812.9.1::pDAB8264.44.06.1 seed wereplanted and grown to maturity. Mature plants were sprayed with a singleherbicide application which consisted of a combination of 2,4-D andglyphosate. The resulting tolerance to these herbicides was measured bycounting the number of surviving plants. Comparatively, control plantswhich did not contain the aad-12 and 2mEPSPS genes and were expected tobe susceptible to the application of the 2,4-D and glyphosate herbicideswere included in the study.

During the first season, herbicide tolerance was assessed in 120 fieldgrown plots of F2 segregating lineages of the breeding stack of soybeanevent pDAB9582.812.9.1::pDAB8264.44.06.1. Each plot was 1 row wide androws were spaced 30 inches apart. Plots were planted on 12 foot centers(total planted length 7.5 feet) with a 4.5 foot alley between plots. Atotal of 4,364 plants from F2 segregating lineages of the breeding stackof soybean event pDAB9582.812.9.1::pDAB8264.44.06.1 were sprayed with amixture of 2,4-D and glyphosate (1120 g ae/ha). A single sprayapplication of the glyphosate/2,4-D herbicides was made between V3 andV4 growth stages. The V3 growth stage is characterized by the unifoliateand first three trifoliate leaves being fully developed and the V4growth stage is characterized by the unifoliate and first fourtrifoliate leaves being fully developed. After the herbicide treatmentwas completed, the plots were observed and 3,234 plants were identifiedas being tolerant to the application of the herbicides. The soybeanevent pDAB9582.812.9.1::pDAB8264.44.06.1 plants which were susceptibleto the herbicide application did not contain copies of the aad-12 and2mEPSPS as a result of Mendelian segregation of the pDAB8264.44.06.1integration event.

During the second season, herbicide tolerance was assessed in greenhousegrown F3 homozygous plants of soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1. The soybean plants were grown in 4inch pots which contained one plant per pot. A total of 15, F3homozygous plants were sprayed with a single application of 2,4-D andglyphosate (840 ae/ha). All 15 plants survived after being sprayed withthe herbicides, indicating that the soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 plants were tolerant to theapplication of the herbicides, glyphosate and 2,4-D.

In summary, the aad-12 and 2mEPSPS genes which were present in thesoybean event pDAB8264.44.06.1 parental line conferred tolerance to2,4-D and glyphosate herbicides. These traits were passed and inheritedin soybean event pDAB9582.812.9.1::pDAB8264.44.06.1, thereby providingherbicidal tolerance to soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1. Comparatively, control plants whichdid not contain the aad-12 and 2mEPSPS genes were susceptible to theapplication of the 2,4-D and glyphosate herbicides.

Example 8.5: Characterization of Insecticidal Activity of Soybean EventpDAB9582.812.9.1::pDAB8264.44.06.1

Greenhouse evaluations were conducted to characterize the insecticidaltolerance of the breeding stack of soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 which resulted from the expression ofthe cry1Ac and cry1F transgenes. Soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 was tested against lab reared soybeanpests including Anticarsia gemmatalis (velvetbean caterpillar) andPseudoplusia includens (soybean looper). The breeding stack of soybeanevent pDAB9582.812.9.1::pDAB8264.44.06.1 was compared against theparental soybean events (soybean event pDAB9582.812.9.1 and soybeanevent pDAB8264.44.06.1) in addition to the non-transformed soybeanvariety Maverick. This comparison was made to determine whether thelevel of plant protection provided by the Cry1F and Cry1Ac proteinswould be present in the breeding stack which introduced additionaltransgenes into the genome of the soybean plant. In addition, thebreeding stack of soybean event pDAB9582.812.9.1::pDAB8264.44.06.1 andsoybean event pDAB8264.44.06.1 were both sprayed with a single herbicideapplication containing 2,4-D and glyphosate (840 g ae/ha) prior to theinsect bioassay to determine whether the spraying of the herbicides hadany effect on the plant protection from insects provided by the Cry1Fand Cry1Ac proteins.

Greenhouse trials were conducted on approximately three week old plants.Ten plants each were used to evaluate the breeding stack of soybeanevent pDAB9582.812.9.1::pDAB8264.44.06.1, soybean eventpDAB9582.812.9.1, and the negative controls; herbicide sprayed soybeanevent pDAB8264.44.06.1 and Maverick. For each insect species tested(Anticarsia gemmatalis and Chrysodeixis (formerly Pseudoplusia)includens), 3 leaf punches were made from each plant for a total of 30leaf discs/plant/insect species. The 1.4 cm diameter (or 1.54 cm²) leafpunches were placed in a test arena on top of 2% water agar, infestedwith one neonate larvae and sealed with a perforated plastic lid.Mortality and leaf consumption were rated four days after infestation.Larvae that were not responsive to gentle probing were considered dead.Leaf damage was assessed by visually scoring the percentage of leafpunch consumed by the insect. Statistical analysis was performed on thedata using JMP® Pro 9.0.1 (2010 SAS Institute Inc., Cary, N.C.).

The results (Table 13) obtained from these replicated experimentsindicated that the level of insect protection and mortality provided bythe Cry1F and Cry1Ac proteins of the breeding stack of soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 were consistent with the parentalsoybean event pDAB9582.812.9.1. As expected, soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 sustained significantly lower insectdamage (0.10-0.15%) than soybean event pDAB8264.44.06.1 (58-76%) and theMaverick (79-91%) control plants for all insects tested. Additionally,high insect mortality (100%) was recorded for all soybean events whichcontained the cry1F and cry1Ac coding sequences, while the negativecontrols, Maverick and soybean event pDAB8264.44.06.1 resulted in <10%insect mortality. Thus, the soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 provided protection from insecticidalactivity at levels comparable to the parental soybean eventpDAB9582.812.9.1.

TABLE 13 Shows the mean percent leaf damage and mortality ofPseudoplusia includens (SBL) and Anticarsia gemmatalis (VBC) fed onvarious soybean events. Mean % Mean % Soybean events Insects leaf damagemortality Maverick SBL 91.46 4.2 VBC 78.96 0 pDAB8264.44.06.1 SBL 75.830 VBC 58.33 8.3 pDAB9582.812.9.1 SBL 0.10 100 VBC 0.15 100pDAB9582.812.9.1 × SBL 0.10 100 pDAB8264.44.06.1 VBC 0.10 100 (n = 24)

Example 9: Breeding Stack of Soybean Event pDAB8264.44.06.1 and SoybeanInsect Tolerant Event pDAB9582.814.19.1 Example 9.1: Sexual Crossing ofSoybean Event pDAB8264.44.06.1 and Soybean Insect Tolerant EventpDAB9582.814.19.1

Soybean event pDAB8264.44.06.1 was sexually crossed with soybean eventpDAB9582.814.19.1. The anthers of soybean event pDAB8264.44.06.1 weremanually rubbed across the stigma of soybean event pDAB9582.814.19.1,thereby fertilizing soybean event pDAB9582.814.19.1. The resulting F1progeny which contained integration events from both soybean eventpDAB9582.814.19.1 and soybean event pDAB8264.44.06.1 were screened fortolerance to 2,4-D and glyphosate herbicides to identify progeny plantswhich contained both integration events. Next, the F1 progeny plantswere self-fertilized to produce an F2 offspring which was confirmed tosegregate independently for both events. The F2 plants were sprayed witha single herbicide application containing both 2,4-D (840 g ae/ha) andglyphosate (840 g ae/ha). The resulting F2 plants were screened using aTaqman zygosity based assay to identify plants that were homozygous forboth events. Selfing of these F2 homozygous plants produced an F3offspring that were homozygous for both soybean event pDAB9582.814.19.1and soybean event pDAB8264.44.06.1. The resulting event was labeled assoybean event pDAB9582.814.19.1::pDAB8264.44.06.1.

Example 9.2: Determination of the Zygosity Status of Soybean EventpDAB9582.814.19.1::pDAB8264.44.06.1

To determine the zygosity status of plants produced from the breedingcross of soybean event pDAB8264.44.06.1 and soybean eventpDAB9582.814.19.1, separate event specific TAQMAN® assays were developedto detect the presence of either the pDAB9582.814.19.1 orpDAB8264.44.06.1 integration events. Segregating F2 plants, producedfrom the self fertilization of a breeding cross of soybean eventpDAB9582.814.19.1 and soybean event pDAB8264.44.06.1, were tested withthese event specific TAQMAN® assays to identify individual plants whichcontained both soybean event pDAB9582.814.19.1 and soybean eventpDAB8264.44.06.1, and were homozygous for both events.

gDNA Isolation

gDNA samples from segregating F2 plants of the breeding stack of soybeanevent pDAB9582.814.19.1::pDAB8264.44.06.1 were tested in this study.Fresh soybean leaf discs, 4 per plant, were collected from 37segregating F2 plants of the breeding stack of soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1. Genomic DNA was extracted fromthese samples using a modified Qiagen MagAttract Plant DNA Kit® (Qiagen,Valencia, Calif.).

TAQMAN® Assay and Results

TAQMAN® primers and probes as previously described were designed for theuse of individual event specific assays for soybean eventspDAB9582.814.19.1 (U.S. Provisional Application No. 61/471,845) andpDAB8264.44.06.1 (described above). These reagents were used with theconditions listed below to determine the zygosity of each integrationevent contained within the breeding stack of soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1.

The multiplex PCR conditions for amplification are as follows: 1× RochePCR Buffer, 0.4 μM event pDAB8264.44.06.1 specific forward primer, 0.4μM event pDAB8264.44.06.1 specific reverse primer 0.4 μM eventpDAB9582.814.19.1 specific forward primer, 0.4 μM eventpDAB9582.814.19.1 specific reverse primer, 0.4 μM Primer GMS116 F, 0.4μM Primer GMS116 R, 0.2 μM Event pDAB9582.814.19.1 specific probe, 0.2μM Event pDAB8264.44.06.1 specific probe, 0.2 μM GMS116 Probe, 0.1% PVP,20 ng gDNA in a total reaction of 10 μl. The cocktail was amplifiedusing the following conditions: i) 95° C. for 10 min., ii) 95° C. for 10sec, iii) 60° C. for 30 sec, iv) 72° C. for 1 sec v) repeat step ii-ivfor 35 cycles, v) 40° C. hold. The Real time PCR was carried out on theRoche LightCycler® 480. Data analysis was based on measurement of thecrossing point (Cp value) determined by LightCycler® 480 software, whichis the PCR cycle number when the rate of change in fluorescence reachesits maximum.

A total of 37 segregating F2 plants, produced from the breeding cross ofsoybean event pDAB9582.814.19.1 and soybean event pDAB8264.44.06.1 weretested with the event specific TAQMAN® assays to determine the zygosityof individual plants for both soybean event pDAB9582.814.19.1 andsoybean event pDAB8264.44.06.1. The results from these assays indicatedthat soybean event pDAB9582.814.19.1 and soybean event pDAB8264.44.06.1were both present and detected in 23 plants. The zygosity status (alsodescribed as ploidy level) of each integration event is indicated inTable 14. Of the 23 identified plants, 1 plant was identified whichcontained two copies of soybean event pDAB9582.814.19.1 and soybeanevent pDAB8264.44.06.1.

TABLE 14 Event specific TAQMAN ® zygosity analysis of the breeding stackof soybean event pDAB9582.814.19.1::pDAB8264.44.06.1 Zygosity status forNumber pDAB9582.814.19.1::pDAB8264.44.06.1 of plantsHomozygous::Homozygous 1 Homozygous::Hemizygous 7 Homozygous::Null 1Hemizygous::Homozygous 3 Hemizygous::Hemizygous 12 Hemizygous::Null 5Null::Homozygous 0 Null::Hemizygous 2 Null::Null 6

Example 9.3: Characterization of Protein Expression in the BreedingStack of Soybean Event pDAB9582.814.19.1::pDAB8264.44.06.1

The biochemical properties of the recombinant Cry1F, Cry1Ac, AAD12,2mEPSPS, and PAT proteins expressed in the breeding stack of soybeanevent pDAB9582.814.19.1::pDAB8264.44.06.1 were characterized. An EnzymeLinked Immunosorbent Assay (ELISA) was used to quantify the expressionof PAT. Comparatively, Cry1Ac/Cry1F and AAD12/2mEPSPS proteins werequantified by multiplexed immunoassays utilizing electrochemiluminescenttechnology from Meso-Scale Discovery (MSD, Gaithersburg, Md.).Collectively, these assays were used to characterize the biochemicalproperties and confirm the robust expression of these proteins in thebreeding stack of soybean event pDAB9582.814.19.1::pDAB8264.44.06.1.

Expression of the PAT Protein in Plant Tissues

Levels of PAT protein were determined in the breeding stack of F3soybean event pDAB9582.814.19.1::pDAB8264.44.06.1 which were identifiedto be homozygous for both event pDAB9582.814.19.1 and eventpDAB8264.44.06.1 integrations. The levels of PAT protein expressed fromsoybean event pDAB9582.814.19.1::pDAB8264.44.06.1 was compared to theparental events, soybean event pDAB9582.814.19.1 and soybean eventpDAB8264.44.06.1.

The soluble, extractable PAT protein was obtained from soybean leaftissue and measured using a quantitative ELISA method (APS 014,Envirologix, Portland, Me.). Samples of soybean leaf tissues wereisolated from greenhouse grown test plants at the unifoliate to V1 stageand prepared for expression analysis. The PAT protein was extracted fromsoybean plant tissues with a phosphate buffered saline solutioncontaining the detergent Tween-20 (PBST) and 1% polyvinylpyrrolidone 40(PVP-40). The samples were then extracted using a GenoGrinder® at 1500rpm for 5 minutes. The plant extract was centrifuged; the aqueoussupernatant was collected, diluted with appropriate buffer as necessary,and analyzed using the PAT ELISA kit in a sandwich format. The kit wasused following the manufacturer's suggested protocol (Envirologix,Portland, Me.).

Detection analysis was performed to investigate the expression andheritability of soybean event pDAB9582.814.19.1::pDAB8264.44.06.1. TheF3 generation of the breeding stack, soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1 expressed PAT at higherconcentrations than either the parental events, pDAB9582.814.19.1 andpDAB8264.44.06.1. The increased concentration of PAT in soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1 breeding stack was expected. Thehigher concentrations of PAT are a result of soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1 containing twice as many copies ofthe pat coding sequence as compared to either of the parental events(Table 15).

TABLE 15 Average PAT protein expression from soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1, and parental events (soybean eventpDAB9582.814.19.1 and soybean event pDAB8264.44.06.1). Average PATExpression Soybean Event (ng/cm²) pDAB9582.814.19.1::pDAB8264.44.06.120.1 pDAB9582.814.19.1 12.0 pDAB8264.44.06.1 13.3

Expression of the Cry1F and Cry1Ac Proteins in Plant Tissues

Levels of Cry1F and Cry1Ac protein were determined in the breeding stackof F3 soybean event pDAB9582.814.19.1::pDAB8264.44.06.1 which wereidentified to be homozygous for both event pDAB9582.814.19.1 and eventpDAB8264.44.06.1 integrations. The levels of Cry1F and Cry1Ac proteinexpressed from soybean event pDAB9582.814.19.1::pDAB8264.44.06.1 wascompared to the parental event, soybean event pDAB9582.814.19.1.

The soluble, extractable Cry1F and Cry1Ac protein was obtained fromsoybean leaf tissue and measured using a multiplexedelectrochemiluminescent MSD assay. Samples of soybean leaf tissue wereisolated from greenhouse grown plants at the unifoliate to V1 stage andprepared for expression analysis. The Cry1F and Cry1Ac protein wasextracted from soybean plant tissues with a phosphate buffered salinesolution containing the detergent Tween-20 (PBST) and 1%polyvinylpyrrolidone 40 (PVP-40). The samples were then extracted usinga GenoGrinder® at 1500 rpm for 5 minutes. The plant extract wascentrifuged; the aqueous supernatant was collected, diluted withappropriate buffer as necessary, and analyzed using a Cry1F/Cry1Acmultiplex MSD assay from Meso-Scale Discovery. The kit was usedfollowing the manufacturer's suggested protocol.

Detection analysis was performed to investigate the expression andheritability of soybean event pDAB9582.814.19.1::pDAB8264.44.06.1. TheF3 generation of the breeding stack of soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1 expressed Cry1Ac protein atconcentrations higher than the parental soybean event pDAB9582.814.19.1.The F3 generation of the breeding stack of soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1 expressed Cry1F protein atconcentrations lower than the parental soybean event pDAB9582.814.19.1.(Table 16). Despite the variability in expression levels, these resultsindicate that soybean event pDAB9582.814.19.1::pDAB8264.44.06.1 plantscontained a functionally expressing copy of the cry1F and cry1Ac codingsequences which were inherited from the parental line, soybean eventpDAB9582.814.19.1.

TABLE 16 Average Cry1Ac and Cry1F protein expression from soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1 as compared to parental eventpDAB9582.814.19.1 Average Average Cry1Ac Cry1F Expression ExpressionSoybean Event (ng/cm²) (ng/cm²) pDAB9582.814.19.1::pDAB8264.44.06.1 25.355.7 pDAB9582.814.19.1 22.4 106.7Expression of the AAD12 and 2mEPSPS Proteins in Plant Tissues

Levels of AAD12 and 2mEPSPS protein were determined in the breedingstack of F3 soybean event pDAB9582.814.19.1::pDAB8264.44.06.1 which wereidentified to be homozygous for both event pDAB9582.814.19.1 and eventpDAB8264.44.06.1 integrations. The levels of AAD12 and 2mEPSPS proteinexpressed from soybean event pDAB9582.814.19.1::pDAB8264.44.06.1 wascompared to the parental event, Soybean Event pDAB8264.44.06.1.

The soluble, extractable AAD12 and 2mEPSPS protein was obtained fromsoybean leaf tissue and measured using a multiplexedelectrochemiluminescent MSD assay. Samples of soybean leaf tissue wereisolated from greenhouse grown plants at the unifoliate to V1 stage andprepared for expression analysis. The AAD12 and 2mEPSPS protein wasextracted from soybean plant tissues with a phosphate buffered salinesolution containing the detergent Tween-20 (PBST) and 1%polyvinylpyrrolidone 40 (PVP-40). The samples were then extracted usinga GenoGrinder® at 1500 rpm for 5 minutes. The plant extract wascentrifuged; the aqueous supernatant was collected, diluted withappropriate buffer as necessary, and analyzed using a AAD12 and 2mEPSPSmultiplex MSD assay from Meso-Scale Discovery. The kit was usedfollowing the manufacturer's suggested protocol.

Detection analysis was performed to investigate the expression andheritability of soybean event pDAB9582.814.19.1::pDAB8264.44.06.1. TheF3 generation of the breeding stack of soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1 expressed AAD12 and 2mEPSPS proteinsat concentrations lower than the parental soybean eventpDAB8264.44.06.1. (Table 17). Despite the variability in expressionlevels, these results indicated that soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1 plants contained a functionallyexpressing copy of the aad-12 and 2mEPSPS coding sequences which wereinherited from the parental line, soybean event pDAB8264.44.06.1.

TABLE 17 Average AAD12 and 2mEPSPS protein expression from soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1 as compared to parental soybeanevent pDAB8264.44.06.1 Average Average AAD12 2mEPSPS ExpressionExpression Soybean Event (ng/cm²) (ng/cm²)pDAB9582.814.19.1::pDAB8264.44.06.1 261.3 127.9 pDAB8264.44.06.1 320.4328.9

Example 9.4: Herbicide Tolerance of the Breeding Stack of Soybean EventpDAB9582.814.19.1::pDAB8264.44.06.1

Herbicide tolerance of the breeding stack, soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1 was assayed. Soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1 seed were planted in a greenhousestudy and mature plants were sprayed with a single herbicide applicationwhich consisted of a combination of 2,4-D and glyphosate. The resultingtolerance to these herbicides was measured by counting the number ofsurviving plants. Comparatively, control plants which did not containthe aad-12 and 2mEPSPS genes and were expected to be susceptible to theapplication of the 2,4-D and glyphosate herbicides were included in thestudy.

Herbicide tolerance was assessed in greenhouse grown F2 plants ofsoybean event pDAB9582.814.19.1::pDAB8264.44.06.1. The soybean plantswere grown in 4 inch pots which contained one plant per pot. A total of37, F3 homozygous plants were sprayed with a single application of 2,4-Dand glyphosate (840 ae/ha) at the unfoliate growth stage. All 25 plantssurvived after being sprayed with the herbicides, indicating that thesoybean event pDAB9582.814.19.1::pDAB8264.44.06.1 plants were tolerantto the application of the herbicides, glyphosate and 2,4-D.

In summary, the aad-12 and 2mEPSPS genes which were present in thesoybean event pDAB8264.44.06.1 parental line conferred tolerance to2,4-D and glyphosate herbicides. These traits were passed and inheritedin soybean event pDAB9582.814.19.1::pDAB8264.44.06.1, thereby providingherbicidal tolerance to soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1. The soybean eventpDAB9582.812.9.1::pDAB8264.44.06.1 plants which were susceptible to theherbicide application did not contain copies of the aad-12 and 2mEPSPSas a result of Mendelian segregation of the pDAB8264.44.06.1 integrationevent. Additionally, control plants which did not contain the aad-12 and2mEPSPS genes were susceptible to the application of the 2,4-D andglyphosate herbicides.

Example 9.5: Characterization of Insecticidal Activity of Soybean EventpDAB9582.814.19.1::pDAB8264.44.06.1

Greenhouse evaluations were conducted to characterize the insecticidaltolerance activity of soybean event pDAB9582.814.19.1::pDAB8264.44.06.1which resulted from the expression of the Cry1Ac and Cry1F transgenes.Soybean event pDAB9582.814.19.1::pDAB8264.44.06.1 was tested against labreared soybean pests including Anticarsia gemmatalis (velvetbeancaterpillar) and Pseudoplusia includens (soybean looper). The breedingstack of soybean event pDAB9582.814.19.1::pDAB8264.44.06.1 was comparedagainst the parental soybean events (soybean event pDAB9582.814.19.1 andsoybean event pDAB8264.44.06.1) in addition to the non-transformedsoybean variety Maverick. This comparison was made to determine whetherthe level of plant protection to insect damage provided by the Cry1F andCry1Ac proteins would be present in the breeding stack which introducedadditional transgenes into the genome of the soybean plant. In addition,the breeding stack of soybean event pDAB9582.814.19.1::pDAB8264.44.06.1and soybean event pDAB8264.44.06.1 were both sprayed with a singleherbicide application containing 2,4-D and glyphosate (840 g ae/ha)prior to the insect bioassay to determine whether the spraying of theherbicides had any effect on the plant protection from insects providedby the Cry1F and Cry1Ac proteins.

Greenhouse trials were conducted on approximately three week old plants.Ten plants each were used to evaluate the breeding stack of soybeanevent pDAB9582.814.19.1::pDAB8264.44.06.1, soybean eventpDAB9582.814.19.1, and the negative controls; herbicide sprayed soybeanevent pDAB8264.44.06.1 and Maverick. For each insect species tested(Anticarsia gemmatalis and Pseudoplusia includens), 3 leaf punches weremade from each plant for a total of 30 leaf discs/plant/insect species.The 1.4 cm diameter (or 1.54 cm²) leaf punches were placed in a testarena on top of 2% water agar, infested with one neonate larvae andsealed with a perforated plastic lid. Mortality and leaf consumptionwere rated 4 days after infestation. Larvae that were not responsive togentle probing were considered dead. Leaf damage was assessed byvisually scoring the percentage of leaf punch consumed by the insect.Statistical analysis was performed on the data using JMP® Pro 9.0.1(2010 SAS Institute Inc., Cary, N.C.).

The results (Table 18) obtained from these replicated experimentsindicated that the level of insect damage and mortality provided by theCry1F and Cry1Ac proteins of the breeding stack of soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1 were consistent with the parentalsoybean event pDAB9582.814.19.1. As expected soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1 sustained significantly lower insectdamage (0.10-0.12%) than soybean event pDAB8264.44.06.1 (58-76%) and theMaverick (79-91%) control plants for all insects tested. Additionally,high insect mortality (100%) was recorded for all soybean events whichcontained the cry1F and cry1Ac coding sequences, while the negativecontrols, maverick and soybean event pDAB8264.44.06.1, resulted in <10%insect mortality. Thus, the soybean eventpDAB9582.814.19.1::pDAB8264.44.06.1 provided protection frominsecticidal activity at levels comparable to the parental soybean eventpDAB9582.814.19.1.

TABLE 18 Shows the mean percent leaf damage and mortality ofPseudoplusia includens (SBL) and Anticarsia gemmatalis (VBC) fed onvarious soybean events. Mean % Mean % Soybean events Insects leaf damagemortality Maverick SBL 91.46 4.2 VBC 78.96 0 pDAB8264.44.06.1 SBL 75.830 VBC 58.33 8.3 pDAB9582.814.19.1 SBL 0.12 100 VBC 0.10 100pDAB9582.814.19.1:: SBL 0.10 100 pDAB8264.44.06.1 VBC 0.10 100 (n = 24)

1.-69. (canceled)
 70. A method of protecting a soybean plant from weedswherein the soybean plant comprises SEQ ID NO:27, said method comprisingapplying a herbicide selected from the group consisting of aphenoxyacetic acid herbicide, a glyphosate herbicide, and a glufosinateherbicide to a field, said field comprising the soybean plant comprisingSEQ ID NO:27.
 71. The method of claim 70, wherein the herbicide is aphenoxyacetic acid herbicide.
 72. The method of claim 71, wherein thephenoxyacetic acid herbicide is 2,4-D.
 73. The method of claim 70,wherein said method comprises applying at least two of said herbicidessimultaneously.
 74. The method of claim 70, wherein said methodcomprises applying at least two of said herbicides sequentially.
 75. Themethod of claim 70, wherein said phenoxyacetic acid herbicide isselected from the group consisting of 2,4-D and 2,4-DB.
 76. The methodof claim 70, wherein said herbicide is applied over the top of saidplant.
 77. A method of breeding a soybean plant, said method comprisingcrossing a first soybean plant comprising SEQ ID NO:27 with a secondsoybean plant to produce a third soybean plant having a genomecomprising SEQ ID NO:27.
 78. The method of claim 77, further comprisingassaying said third soybean plant for the presence of SEQ ID NO:27 insaid genome.
 79. The method of claim 77, wherein said method is used tointrogress a herbicide tolerance trait into the third soybean plant. 80.A method for identifying Event pDAB8264.44.06.1 in a sample, said methodcomprising detecting a junction sequence of Event pDAB8264.44.06.1, aspresent in seed deposited under ATCC Accession number PTA-11336, with aprobe or at least one primer that specifically binds with or amplifiessaid junction sequence, said junction sequence comprising residues561-580 of SEQ ID NO: 14, or residues 211-230 of SEQ ID NO:
 15. 81. Themethod of claim 80, said method further comprising amplifying a DNAfragment from a nucleic acid present in said sample using a polymerasechain reaction with at least a first primer and a second primer, whereinsaid Event pDAB8264.44.06.1 comprises SEQ ID NO:27 and wherein saidfirst primer specifically binds an insert sequence within said SEQ IDNO:27, or the complement thereof, and said second primer specificallybinds a sequence within a flanking sequence of SEQ ID NO:27, or thecomplement thereof, wherein said flanking sequence is selected from thegroup consisting of SEQ ID NO: 1 and SEQ ID NO:2.
 82. A method fordetermining event zygosity of a soybean plant comprising soybean EventpDAB8264.44.06.1, as present in seed deposited under ATCC Accessionnumber PTA-11336, said Event pDAB8264.44.06.1 comprising a transgeneconstruct, said transgene construct being flanked by a 5′ flankingsoybean genomic DNA and a 3′ flanking soybean genomic DNA, said methodcomprising: obtaining a DNA sample comprising genomic DNA from saidsoybean plant; producing a contacted sample by contacting said DNAsample with a. a first event primer and a second event primer, whereinsaid first event primer specifically binds said transgene construct,said second event primer specifically binds said 5′ flanking soybeangenomic DNA or said 3′ flanking soybean genomic DNA, and wherein saidfirst event primer and said second event primer produce an eventamplicon when subjected to TaqMan PCR conditions; b. a reference forwardprimer and a reference reverse primer that produce a reference ampliconfrom an endogenous soybean reference gene when subjected to TaqMan PCRconditions; c. a florescent event probe that hybridizes with said eventamplicon; d. a florescent reference probe that hybridizes with saidreference amplicon; subjecting said contacted sample tofluorescence-based endpoint TaqMan PCR conditions; quantitating saidflorescent event probe that hybridized to said event amplicon;quantitating said florescent reference probe that hybridized to saidreference amplicon; comparing amounts of hybridized florescent eventprobe to amounts of hybridized florescent reference probe; anddetermining zygosity of said Event pDAB8264.44.06.1 by comparingflorescence ratios of said hybridized fluorescent event probe and saidhybridized fluorescent reference probe.
 83. The method of claim 82,wherein said amplicons consist of about 50 to about 150 nucleotideresidues.
 84. The method of claim 82, wherein said 5′ flanking soybeangenomic DNA comprises SEQ ID NO: 1, and said 3′ flanking soybean genomicDNA comprises SEQ ID NO:2.
 85. The method of claim 82, wherein saidreference gene is an endogenous soybean GMFL01-25-J19 gene.
 86. Themethod of claim 82, wherein said first event primer binds an insertsequence of SEQ ID NO:27, and said second event primer binds SEQ ID NO:1or SEQ ID NO:2, or the complements thereof.
 87. The method of claim 82,wherein said method is used for breeding introgression of the EventpDAB8264.44.06.1 into a soybean line.
 88. The method of claim 87,wherein said soybean line lacks said Event pDAB8264.44.06.1.
 89. Themethod of claim 82, wherein said reference gene comprises or hybridizesto a sequence selected from the group consisting of SEQ ID NO:24, SEQ IDNO:25, and SEQ ID NO:26.
 90. The method of claim 82, wherein saidreference primers are selected from the group consisting of SEQ ID NO:25 and SEQ ID NO:24, and said reference probe comprises SEQ ID NO:26.91. The method of claim 82, wherein said fluorescent event probe andsaid fluorescent reference probe are labeled with a fluorescent dye anda quencher.
 92. The method of claim 91, wherein said fluorescent eventprobe comprises FAM as said fluorescent dye at the 5 ‘ end of saidfluorescent event probe and an MGB quencher on the 3’ end of saidfluorescent event probe.
 93. The method of claim 91, wherein saidfluorescent reference probe is labeled with HEX at the 5′ end of saidfluorescent reference probe and a Black Hole Quencher 1 (BHQ1) at the 3′end of said fluorescent reference probe.
 94. The method of claim 82,wherein said fluorescent event probe comprises SEQ ID NO:20.
 95. Themethod of claim 82, wherein said event primers are selected from thegroup consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:21, and SEQID NO:22.
 96. The method of claim 82, wherein results of said method areread directly in a plate reader.
 97. The method of claim 82, whereinsaid DNA sample is obtained from a soybean plant in a field.
 98. A kitfor performing the method of claim 82, said kit comprising said firstevent primer, said second event primer, said reference forward primer,said reference reverse primer, said event probe, and said referenceprobe.
 99. The kit of claim 98, wherein said first event primer consistsof SEQ ID NO: 18 and said second event primer consists of SEQ ID NO: 19,said reference forward primer consists of SEQ ID NO:24 and saidreference reverse primer consists of SEQ ID NO:25, said event probeconsists of SEQ ID NO:20, and said reference probe consists of SEQ IDNO:26.
 100. A kit for performing the method of claim 80, said kitcomprising said probe and said at least one primer.
 101. Apolynucleotide comprising SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQID NO:16, SEQ ID NO:17, or SEQ ID NO.27.
 102. A polynucleotidecomprising SEQ ID NO:13.
 103. A polynucleotide comprising SEQ ID NO.27.104. A meal product comprising SEQ ID NO:27.
 105. An isolated primer orprobe having the polynucleotide sequence of any of SEQ ID NOS:3 to 12 or18 to
 26. 106. A soybean seed comprising a cell comprising SEQ ID NO:27,said cell further comprising SEQ ID NO:29.
 107. A soybean plant grownfrom the seed of claim 106, said plant comprising said SEQ ID NO:27, andsaid SEQ ID NO:29.
 108. The plant of claim 107 wherein said SEQ ID NO:27provides herbicide tolerance to the plant and said SEQ ID NO:29 providesinsect resistance to the plant.
 109. A part of the plant of claim 108wherein said part is selected from the group consisting of an ovule, aflower, a shoot, a root, and a leaf, said part comprising SEQ ID NO:27and SEQ ID NO:29.
 110. An expression cassette inserted transgenicallyinto a single chromosomal locus of the genome of a soybean plant cell toproduce said genome comprising SEQ ID NO:27.
 111. The expressioncassette of claim 110, wherein said SEQ ID NO: 27 comprises a firstplant transcription unit which expresses a glyphosate herbicidetolerance gene, a second plant transcription unit which expresses aphenoxyacetic acid herbicide tolerance gene, and a third planttranscription unit which expresses a glufosinate herbicide tolerancegene.